quality evaluation of algerian honeys: eucalyptus, jujube

Dissertation submitted to Escola Superior Agrária de Bragançca to obtain the

Degree of Master in Biotechnological engineering under the scope of the double

diploma with Université Moulay Taher in Algeria

Seloua Kaid

Supervised by

Miguel José Rodrigues Vilas Boas

Soraia Isabel Domingues Marcos Falcão

Kaddour Ziani

Bragança 2021

Quality evaluation of Algerian honeys:

Eucalyptus, Jujube, Euphorbia and multiflora

I

Acknowledgment

First of all, I want to thank Professor Miguel Vilas Boas, for having dragged me to this great

school, this great institution (IPB), for all the knowledge he transmitted to me, for his patience,

dedication, permanent availability, support, advice, for friendship and good mood. Thank you

very much, for the topic you proposed to develop, which made me interested in the world of

bees.

Also want to thank my co-supervisor Dr. Soraia Falcão, for her wise advice and suggestions,

for her patience and for the support provided at all times of this work.

I am grateful to my co-supervisor, Dr Kaddour Ziani, for all his commitment, professionalism,

understanding, patience and support shown throughout this work.

I would also like to thank Professor Paulo Russo-Almeida from LabApisUTAD

, for its

collaboration in the melissopalynological analysis

I also thank my dear colleague Andreia Tomás for the availability shown in the laboratory and

for the transmission of knowledge, for companionship, help, guidance, patience, advice,

courage, good disposition, friendship and understanding demonstrated throughout the work.

Thank you very much!

I would like to thank the professors of the Master in biotechnological engineering, from the

Escola Superior Agraária de Bragança, my thanks for the knowledge that was transmitted to

me over this year and for the push they gave me to make this step a reality.

To the entire CIMO laboratory team that were always available to assist in whatever was

necessary, for having integrated me and for the knowledge they transmitted to me.

I also think the biochemistry department at the Faculty of Science of the University of Dr

moulay Taher University Saida Algeria.

II

DEDICATION

This thesis is dedicated to:

The sake of Allah, my Creator and my Master

My great teacher and messenger, Mohammed (May Allah bless and grant him), who

taught us the purpose of life.

My homeland Algeria, the warmest womb that I miss it a lot, and I am just waiting

impatiently to breathe its fresh air again after a year and six months absence

My great parents, who never stop giving of themselves in countless ways, I miss you

so much and forgive me for the long absence

My beloved brothers and sisters

My friends who encourage and support me

All the people in my life who touch my heart, I dedicate this research.

III

Abstract

This study was intended to evaluate the quality parameters of ten honey samples, from

various regions in semi-arid region of Algeria. Different parameters such as the

melissopalynological and the physicochemical properties of the honeys (moisture, color,

electrical conductivity, 5-hydroxymethylfurfural, pH, acidity, proline, and diastase activity)

were determined, as well as the evaluation of sugars, minerals and phenolic compounds.

Nutritional composition, antioxidant activity (reducing power and DPPH free radical

scavenging activity), anti-inflammatory and cytotoxicity were also evaluated. Finally,

antibiotics residues such as sulphonamides and tetracyclines antibiotics residues were

screened.

The melissopalynological results identified ten types of pollen, with Cytisus striatus

pollen being identified as the most abundant, present in all samples with percentages between

26.0 % and 83.8 %. EC1, MF1 and MF2 (Sidi Belabes region) were classified as monofloral

of Cytisus striatus honey. Additionally, although samples J1 to J3 were not considered as

Jujube monofloral, they showed a high percentage of Ziziphus pollen. The remaining samples

(EC2, EF1, EF2, and EF3) were classified as multifloral.

Regarding the physicochemical parameters, no significant differences were found in

the color of the samples which ranged between amber, light amber and extra light amber.

Moisture content was found to be between 13.6% (EF1) and 18.3% (EC1), while pH values

ranged between 4.2 and 5.1. Electrical conductivity values varied between 270 and 410

μS.cm-1

, while 5- hydroxymethylfurfural content showed values between 0 and 36.5 mg.kg

-1

and diastase values between 8.8 DN and 13.3 DN. Concerning the proline content, the

samples showed proline levels between 2.2–4.7 mg/kg, indicating a good maturity of the

honeys and absence of adulteration. All the honeys meet the standard required by the

European legislation with exception of the diastase index. The sugar profile, analyzed by high

pressure liquid chromatography with refractive index detection (HPLC-RI), showed that all

samples have higher fructose content than glucose, being the total more than 88.70 %,

allowing the classification of all the samples as nectar honeys.

Within the minerals, potassium was quantitatively the most important mineral (72.93%

of total minerals quantified), having an average content 730.59mg/kg, followed by sodium,

calcium and magnesium, with 17.05%, 4.43% and 4.22%, respectively, while cadmium and

lead had the lowest concentration, 0.003 % and 0.04% respectively.

IV

The total phenolic content of the analyzed honey samples ranged between 0.7 mg

GAE/g, for samples EF and J and 1.4 mg GAE/g, for samples EC, with an average of 0.9 mg

GAE/g. The total flavonoid content varied from 0.03 to 0.09 mg QE/g with the highest levels

observed in J honey samples. The values obtained for DPPH ranged from 0.02 to 0.04 mg/mL,

without significant differences between the samples.

The analysis of the phenolic profile was performed by UPLC/DAD/ESI-MSn, where

nineteen phenolic compounds were identified, including six phenolic acids, nine flavonoids,

two isoprenoids (abscisic acid isomers), one phenolic diterpenoid (carnosol) and one

spermidine (N1, N

5, N

10-tri-p-coumaroyespermidine). The major quantity of phenolic

compounds was found in sample EC1 with 202 mg/100 g, while sample EF3 showed the

lowest amount with 59.85 mg/100 g.

Concerning the anti-tumoral evaluation, all the studied extracts presented good activity,

with MF1 showing the highest cytotoxicity, followed by EF1. Also, all the extracts under

study showed anti-inflammatory capacity, with IC50 values between 8 and 400 µg/mL.

Regarding the antibiotics residues, its presence was found in three of the samples

(MF1 EF1 EF3) showed positive results for sulphonamides residues.

Keywords: honey, melissopalynological analysis, physicochemical parameters, antioxidant

activity, anti-inflammatory activity, cytotoxicity, antibiotic residues

V

Resumo

Este estudo teve por objetivo avaliar os parâmetros de qualidade de dez amostras de mel, de

várias regiões da região semiárida da Argélia. Neste âmbito foram determinadas as

características melissopalinológicas e os parâmetros físico-químicos dos méis (humidade, cor,

condutividade elétrica, 5-hidroximetilfurfural, pH, acidez, prolina e diástase), bem como

efetuada a avaliação do perfil de açúcares, minerais e compostos fenólicos. A presença de

resíduos de antibióticos como sulfonamidas e tetraciclinas foi também verificada.

Paralelamente foi estudada a composição nutricional dos méis e a sua bioatividade através da

atividade antioxidante (DPPH e poder redutor), anti-inflamatória e citotoxicidade.

Os resultados melissopalinológicos identificaram dez tipos de pólen, sendo o pólen de

Cytisus striatus o mais frequente, estando presente em todas as amostras com percentagens

entre 26,0% e 83,8%. As amostras EC1, MF1 e MF2 (região de Sidi Belabes) foram

classificados como méis monoflorais de Cytisus striatus. Já as amostras J1, J2 e J3, não

tenham sido consideradas monoflorais de Jujube, apresentaram uma alta percentagem de

pólen de Ziziphus. As restantes amostras (EC2, EF1, EF2 e EF3) foram classificadas como

méis multiflorais.

Em relação aos parâmetros físico-químicos, não foram encontradas diferenças significativas

na cor das amostras que variaram entre âmbar, âmbar claro e âmbar extra claro. Os resultados

do teor de humidade encontrados ficaram entre 13,6% (EF1) e 18,3% (EC1), enquanto os

valores do pH variaram entre 4,2 e 5,1. Os valores da condutividade elétrica variaram entre

270 e 410 μS.cm-1

, enquanto o conteúdo de 5-hidroximetilfurfural apresentou valores entre 0

e 36,5 mg.kg-1

e a diástase variou entre 8,8 DN e 13,3 DN. Quanto ao conteúdo de prolina, as

amostras apresentaram níveis de prolina entre 2,2–4,7 mg/kg, indicando boa maturidade dos

méis e ausência de adulteração. Todos os méis presentaram valores dentro do requerido pela

legislação europeia, com exceção do índice de diástase. O perfil de açúcares, analisado por

cromatografia líquida de alta pressão com deteção de índice de refração (HPLC-RI),

confirmou um maior teor de frutose do que glucose, sendo o total superior a 88,7%,

permitindo a classificação de todas as amostras como méis de néctar.

O potássio foi o mineral encontrado em maior quantidade (72,93% dos minerais totais

quantificados), tendo um teor médio de 730,59mg/kg, seguido do sódio, cálcio e magnésio

com17,05%, 4,43% e 4,22% respetivamente), enquanto o cádmio e o chumbo apresentaram a

concentração mais baixa, 0,003% e 0,04%, respetivamente.

VI

O conteúdo fenólico total das amostras variou entre 0,7 mg GAE/g, para as amostras EF e J

e 1,4 mg GAE/g, para as amostras CE, apresentando uma média de 0,9 mg GAE/g. O teor de

flavonóides totais variou entre 0,03 e 0,09 mg QE/g, sendo as amostras J as que apresentaram

um valor mais elevado. Os valores obtidos para o DPPH variaram entre 0,02 e 0,04 mg/mL,

sem diferenças significativas entre as amostras.

A análise do perfil dos compostos fenólicos foi realizada por UPLC/DAD/ESI-MSn, onde

foram identificados dezanove compostos fenólicos, incluindo seis ácidos fenólicos, nove

flavonóides, dois isoprenóides (isómeros do ácido abscísico), um diterpenóide fenólico

(carnosol) e uma espermidina (N1, N

5, N

10-tri-p-coumaroyespermidina). A amostra EC1

apresentou a maior quantidade de compostos fenólicos com 202 mg/100g, enquanto a amostra

EF3 apresentou a menor quantidade com 59,85 mg/100 g.

Quanto à avaliação anti-tumoral, todos os extratos estudados apresentaram atividade, sendo

o MF1 o que apresentou maior citotoxicidade, seguido do EF1. Além disso, os extratos

apresentaram capacidade anti-inflamatória, com valores de IC50 entre 8 e 400 µg/mL.

Em relação aos resíduos de antibióticos verificou-se a presença de três das amostras (MF1,

EF1, EF3) com resultados positivos para resíduos de sulfonamidas.

Palavras-chave: mel, análise melissopalinológica, parâmetros físico-químicos, atividade

antioxidante, atividade anti-inflamatória, citotoxicidade, resíduos de antibióticos

VII

Index

Acknowledgment .................................................................................. I

Abstract .............................................................................................. III

Resumo ................................................................................................. V

Figures Index ....................................................................................... X

Tables index ....................................................................................... XI

Abbreviations List ........................................................................... XII

Chapter I- Introduction ...................................................................... 1

Introduction .......................................................................................... 2

1.1. Objectives ........................................................................................................................ 2

1.2. Honey bees and bee products .......................................................................................... 4

1.2.1. Apis mellifera ........................................................................................................... 4

1.2.2. Bee Products ............................................................................................................. 5

1.3. Honey categories concerning origin ................................................................................ 6

1.3.1. Nectar honey ............................................................................................................ 6

1.3.2. Honeydew honey ...................................................................................................... 6

1.4. Honey chemical composition .......................................................................................... 7

1.4.1. Sugars ....................................................................................................................... 8

1.4.2. Water content ........................................................................................................... 8

1.4.3. Proteins and amino acids .......................................................................................... 8

1.4.4. Enzymes ................................................................................................................... 9

1.4.5. 5-Hydroxylmethylfurfural (5-HMF) ........................................................................ 9

1.4.6. Organic acids .......................................................................................................... 10

1.4.7. Vitamins ................................................................................................................. 10

1.4.8. Mineral content ...................................................................................................... 10

1.4.9. Volatile compounds ................................................................................................ 11

1.4.10. Phenolic compounds ............................................................................................ 11

1.5. Other physicochemical parameters ............................................................................... 12

1.5.1. Color ....................................................................................................................... 12

1.5.2. Electrical conductivity ............................................................................................ 12

1.5.3. pH and acidity ........................................................................................................ 12

VIII

1.6. Antibiotic residues in honey .......................................................................................... 13

1.7. Biological properties of honey ...................................................................................... 13

1.8. Beekeeping in Algeria ................................................................................................... 14

1.9. Algerian honey .............................................................................................................. 15

1.9.1. Eucalyptus honey ................................................................................................... 15

1.9.2. Euphorbia honey .................................................................................................... 16

1.9.3. Jujube honey ........................................................................................................... 17

Chapter II- Materials and methods ................................................. 18

2.Material and methods .................................................................... 19

2.1. Honey samples .............................................................................................................. 19

2.2. Honey analysis .............................................................................................................. 20

2.2.1. Pollen analysis ........................................................................................................ 20

2.2.2. Physicochemical analysis ....................................................................................... 21

2.2.3. Ash content ............................................................................................................. 29

2.2.3.2. Protein content ..................................................................................................... 29

2.3. Spectrophotometric analysis of the phenolic compounds ............................................. 30

2.3.1. Total phenolic content ................................................................................................ 30

2.3.2Total flavonoid content ................................................................................................ 30

2.4. Phenolic compounds ..................................................................................................... 30

2.4.1. Extraction ............................................................................................................... 30

2.4.2. Phenolic profile by UPLC / DAD / ESI-MSn ......................................................... 31

2.5. Antioxidant activity ....................................................................................................... 33

2.5.1. DPPH˙ assay ........................................................................................................... 33

2.5.2. Reducing power activity ......................................................................................... 34

2.6. Cytotoxic potential ........................................................................................................ 34

2.7. Anti-inflammatory activity ............................................................................................ 35

2.8. Detection of antibiotics residues ................................................................................... 35

2.8.1.Tetracycline residues ............................................................................................... 36

2.8.2. Sulphonamide residues ........................................................................................... 37

Chapter III- Results and discussion ................................................. 38

3. Results and discussion ................................................................... 39

3.1. Melissopalynological analysis ....................................................................................... 39

IX

3.2. Physicochemical parameters ......................................................................................... 40

3.2.1. Color ....................................................................................................................... 40

3.2.2. Moisture content ..................................................................................................... 41

3.2.3. Electrical conductivity ............................................................................................ 42

3.2.4. pH, free, lactonic and total acidity ......................................................................... 43

3.2.5. Proline .................................................................................................................... 44

3.2.6. 5-HMF .................................................................................................................... 45

3.2.7. Diastase activity ..................................................................................................... 46

3.3. Sugar analysis ............................................................................................................ 46

3.4. Minerals ......................................................................................................................... 48

3.5. Nutritional parameters ................................................................................................... 50

3.6. Total phenolics and total flavonoids contents ............................................................... 51

3.7. Phenolic compounds by UPLC / DAD / ESI-MSn ........................................................ 52

3.8. Antioxidant activity ....................................................................................................... 57

3.8.1. DPPH ...................................................................................................................... 57

3.8.2. Reducing power ...................................................................................................... 57

3.9. Cytotoxic potential ........................................................................................................ 58

3.10. Anti-inflammatory activity .......................................................................................... 59

3.11. Screening of antibiotics residues ................................................................................. 60

Chapter IV- Conclusion and Future Perspectives ......................... 62

Conclusion .......................................................................................... 63

Future perspectives ............................................................................ 65

Chapter V- References ...................................................................... 66

References ........................................................................................... 67

Chapter VI- Appendix ....................................................................... 83

Appendix ............................................................................................. 84

X

Figures Index

Figure 1. (A) Worker European honeybee, Apis mellifera Linnaeus. (B) A Queen. (C) Drone

(male) European honeybee, Apis mellifera. Photograph by Alexander wild

https://www.alexanderwild.com/Insects/Stories/Honey-Bees/i-3DtbsJ. .................................... 4

Figure 2. 5-HMF formation resulting from a sugar decomposition reaction (Bogdanov, 2014)

.................................................................................................................................................. 10

Figure 3. (A) The Langstroth hive and (B) the Langstroth hive different parts (John, 2014).

.................................................................................................................................................. 14

Figure 4. Number of honeybee colonies in Algeria from 2002 to 2010. (B) Honey production

in Algeria from 2002 to 2010. Source: Ministry of Agriculture and Rural Development:

MADR (2009-2010) (Adjlane, Doumandji and Haddad N. al., 2012). .................................... 14

Figure 5. Images showing (A): Apis mellifera intermissa bee and a (B): Apis mellifera

Sahariensis bee (Tlemcani, 2013). ........................................................................................... 15

Figure 6. (A) Eucalyptus plant (Orantes, Gonell, Torres et al., 2018). (B) Euphorbia plant. (C)

Jujube plant (Photograph by Andrii Salomatin,

https://www.shutterstock.com/fr/g/Andrii%2BSalomatin retrieved on 24-05-2 ..................... 17

Figure 7. Geographic origin of the honey samples. ................................................................ 19

Figure 8. Conductivity meter. .................................................................................................. 21

Figure 9. Potenciometer titrator. .............................................................................................. 22

Figure 10. Phenolic compounds extraction stages; acidified water (pH 2) (A), deionized water

(B), and methanol (C). .............................................................................................................. 31

Figure 11. UPLC / DAD / ESI-MSn

equipment ...................................................................... 33

Figure 12. Charm LSC 7600 ................................................................................................... 36

XI

Tables index Table 1. Honey composition after (Bogdanov, 2009) values in g/100g. ................................... 7

Table 2. Physicochemical properties of Jujube, Euphorbia, Eucalyptus honeys of arid and

semi-arid zones in north Africa ................................................................................................ 16

Table 3. Geographic origin and other information from honey samples. ................................ 20

Table 4. Calibration curve for sugars ...................................................................................... 24

Table 5. The calibration standards used in the spectrophotometer for the determination of

potassium and sodium. ............................................................................................................. 25


calcium and magnesium. .......................................................................................................... 26

Table 7. The calibration standards used in the spectrophotometer for the determination of iron.

.................................................................................................................................................. 27

Table 8. The calibration standards used in the spectrophotometer for determination of lead . 28


manganese, copper, and cadmium. ........................................................................................... 28

Table 10. DPPH assay steps. ................................................................................................... 33

Table 11. Pollen characteristics of the analyzed honey samples. ............................................ 40

Table 12. Physicochemical parameters: color, moisture content and conductivity. ................ 41

Table 13. pH and acidity of the honey samples analyzed........................................................ 44

Table 14. Physicochemical parameters of honey: 5- HMF, diastase and proline. .................. 46

Table 15. Sugar profile, obtained by HPLC-RI, of the studied honey samples (values

expressed in g/100g of honey). ................................................................................................ 48

Table 16. Minerals contents, obtained by using flame atomic absorption spectrophotometer

(values expressed in mg/100 kg of honey). .............................................................................. 49

Table 17. Nutritional values of honey: Ash, energy, proteins and carbohydrates. .................. 50

Table 18. Total phenolic and total flavonoid contents and antioxidant activity of honey

samples. .................................................................................................................................... 52

Table 19. Phenolic compounds and abscisic acid identified by UPLC/DAD/ESi-MSn in the

honey samples under study. ..................................................................................................... 53

Table 20. Quantification of phenolic compounds, expressed in mg/100 g honey. .................. 56

Table 22. Cytotoxicity potential and anti-inflammatory activity (GI50 values, µg/mL).......... 59

Table 23. Residues screening using CHARM II. .................................................................... 60

XII

Abbreviations List

[M-H]- - Ion product

5-HMF - 5-Hydroxymethylfurfural

Abs – Absorbance

AFB- American foulbrood

DN- Diastase index

GAE- Gallic acid equivalents

EU- European Union

HPLC– High pressure liquid chromatography

IHC– International Honey Commission

IR - Refractive index

LC- Liquid chromatography

LC-MS- Liquid chromatography coupled to mass spectrometry

MS- Mass spectrometry

m/z- mass to charge ratio

QE- quercetin equivalents

rpm- Rotation per minute

SPE- Solid phase extraction

TR - Retention time

UPLC/DAD/ESI-MSn- Ultra-pressure liquid chromatography with photodiode detection

coupled to tandem mass spectrometry with electrospray ionization.

Chapter I- Introduction

1



2

Introduction

Algeria has a rich variety of melliferous plants, which is distributed in different

bioclimatic zones. It has a potentially large beekeeping production area, but honey production

remains low. This weakness is due to the lack of expertise of intensive production techniques

on the part of beekeepers, but also due to climate change and absent of transhumance.

In Algeria, the agricultural sector set up during the year 2000 an operational strategy

for agricultural development (national agricultural development plan PNDA) extended from

2002 to the rural domain in favor of new attributions entrusted by the government to the

ministry of agriculture and rural development. In this context, attention was given to

beekeeping production and in particular to the establishment of modern hives and the

production of honey (Adjlane, Doumandji and Haddad, 2012).

Honey is the world's primary sweetener and nature's original sweetener prepared by

honeybees. Honey has been used as a food and medicine for at least 6000 years. The demand

for high quality honey is attracting great attention because of its health benefits (Alvarez-

Suarez et al., 2010) derived from its diversity and has been shown to have biological

properties, such as antimicrobial, antiviral, antiparasitic, anti-inflammatory, antioxidant,

antimutagenic and antitumor effects (Bogdanov, Jurendic, Sieber, & Gallmann, 2008).

Diseases prevention through consumption of honey is probably due to the presence of more

than 181 substances, such as amino acids, enzymes, proteins, vitamins, minerals, ash, organic

acids and phenolic compounds (Ouchemoukh et al., 2007; Ferreira et al., 2009). Its

composition varies with the floral source used by the bees, the harvest period and the geo-

climatic conditions of the regions concerned (Mbogning et al., 2011). In Algeria, several

studies on honey characterization have been carried out; we can cite the studies of: (Chefrour,

2007), (Ouchemoukh et al, 2007), (Makhloufi et al 2010), (Zerrouk et al 2011), (Zerrouk et al,

2014), (Nair, 2014), (Draiaia et al, 2015) and (Haouam et al, 2016).

1.1. Objectives

Algerian beekeepers who have constantly attempted to rescue and guarantee the

common characteristics of honey hope to discover different markets from local ones. For that,

an extensive study of the Algerian honey is needed, having in mind the establishment of

quality and authenticity guidelines and regulations. The aim of the present study is to evaluate

the quality of Algerian honey and verify its compliance with the established standards of

Codex. For that, ten samples with different botanical and geographical origin were analyzed

https://www.sciencedirect.com/science/article/pii/S1466856413001094#bb0015




3

regarding the following physicochemical parameters: melissopalynological analysis, color,

moisture, acidity, pH, ash content, electrical conductivity, diastase index, proline, 5-

hydroxymethylfurfural (HMF), nutritional composition and mineral content. Phenolic

compounds were evaluated through spectrophotometric methods and liquid chromatography

coupled with mass spectroscopy (LC-MS). Antioxidant activity (reducing power, DPPH free

radical scavenging activity), cytotoxicity and anti-inflammatory activities were also evaluated.

Finally, the presence of antibiotics, recurrent residues in honey, such as tetracyclines and

sulphonamides were screened to attest its safety.


4

1.2. Honey bees and bee products

1.2.1. Apis mellifera

Apis mellifera naturally occurs in Europe, the Middle East, and Africa. This

species has been subdivided into at least 20 recognized subspecies (Mortensen, Schmehl

and Ellis, 2013). Like all Hymenopterans, honeybees have haplo-diploid sex

determination. Unfertilized eggs develop into drones (males), and fertilized eggs develop

into females. Female larvae, which taken care with a standard food regimen of pollen,

nectar, and brood nourishment become grown-up worker bees. Female larvae fed with a

rich food regimen of royal jelly, pollen, and nectar become queen (Mortensen, Schmehl

and Ellis, 2013). Worker honeybees are non-reproductive females. They are the smallest

in physical size of the three ranks and their body is designed specifically for pollen and

nectar collection (Fig.1.A). Queen honeybee (Fig.1.B) is the only reproductive female in

the colony. Her head and thorax are similar in size compared to that of the worker, while

the abdomen is more extended and plumper. Drones are the male cast of honeybees.

Drone's head and thorax are bigger than those of the females, (Fig.1.C) (Mortensen,

Schmehl and Ellis, 2013).

Figure 1. (A) Worker European honeybee, Apis mellifera Linnaeus. (B) A Queen. (C) Drone

(male) European honeybee, Apis mellifera. Photograph by Alexander wild

https://www.alexanderwild.com/Insects/Stories/Honey-Bees/i-3DtbsJ.

A

B

C


5

1.2.2. Bee Products

1.2.2.1. Beeswax

Beeswax is an extremely inert common material that is secreted by worker bees

from the wax glands (Avshalom and Yaacov, 1996). Bees use beeswax to grow their

larvae and construct honeycomb cells where pollen and honey are stored. When secreted

by bees, beeswax is white, but in the honey combs rapidly obscures due to the contact

with the bees and also the pollen and honey (Avshalom and Yaacov, 1996).

1.2.2.2. Propolis

The word propolis comes from the Greek «pro» = in front, «polis» = city, and

means a substance with a protecting role for the bee colony (Bogdanov, 2014). Bees

gathered resinous exudates from leaf buds, shoots and petioles of leaves from different

plants with their mandibles, which once introduced into the hive, are mixed with wax and

salivary secretions, in order to produce propolis, which is used as a building and defense

material within the hive. Propolis has a very complex composition which is dependent on

the plant origin (Bankova and De Castro, 2000). The main chemical classes and most

bioactive compounds found in propolis are the phenolic compounds, which are

responsible for most of the bioactivities (Bankova and De Castro, 2000).

1.2.2.3. Royal jelly

Royal jelly is a bee product secreted by the hypopharyngeal and mandibular glands

of the nurse working bees (Zahran et al., 2016), between the 6th

and 12th

day of their life

cycle. This bee product is a white-yellow colloid with a pH between 3.6–4.2, with a

variable composition which depends on the metabolic and physiologic condition of the

worker bees, bee specie and on the seasonal and local conditions (Scorselli and Donadio,

2005).

1.2.2.4. Bee pollen and bee bread

Pollen grains are microscopic structures, male gametes located in the anthers of

stamens, indispensable for the fertilization of the female sexual organ of the flower (Krell,

1996). Pollen is extremely important for the hive, it is the main source of food for the

larvae providing them with important nutrients for their development such as proteins, and

carbohydrates, lipids, vitamins and minerals (Luz et al.,2010).


6

1.2.2.5. Bee venom

Bee venom (BV) is an odorless and transparent liquid produced by female worker

bees containing a hydrolytic mixture of proteins with acid pH (4.5 to 5.5) that bees often

use as a defense tool against predators. One drop of BV consists of 88% of water and only

0.1 µg of dry venom (Bellik, 2015)

1.2.2.6. Honey

The Codex Alimentarius defined honey as a natural sweet substance, produced by

honeybees from the nectar of plants, secretions of their living parts, or excretions of plant-

sucking insects on the living parts of plants, which the bees collect, transform by

combining with specific substances of their own, deposit, dehydrate, store and leave in

honeycombs to ripen and mature (Codex Alimentarius, 2001). The definition of honey

under European Union (EU) legislation is very similar, with the difference that it

stipulates the bee species as being Apis mellifera (Directive 2001/110/EC).

1.3. Honey categories concerning origin

1.3.1. Nectar honey

This type of honey is produced by bees after they harvest the nectar of the flowers.

Nectar is a sugar-rich liquid produced by plants in glands called nectaries, and mainly

exist to encourage pollination by insects and other animals. About 95% of the dry

substance are sugars, the rest are amino acids (0.05 %), minerals (0.02-0.45 %) and

restricted amounts of organic acids, nutrients, and vitamins (Bogdanov, 2014). According

to their botanical origin, nectar honeys can be classified as monofloral honeys, if they are

produced from a single family or plant species, or as multifloral honeys when there is no

floral species that stands out. This assessment is often carried out through an analysis of

pollen grains that are present in honey, considering that when collecting nectar in the

flower, bees transport pollen grains that they will inadvertently introduce into honey

(Bear, 2009).

1.3.2. Honeydew honey

Honeydew honey is formed from secretions of living parts of plants or from the

excretions of sucking insects (Hemiptera, mostly aphids) (Terrab et al., 2003). These

insects break the plant cell and ingest the sap. The excess is excreted as droplets of

honeydew, which are gathered by the bees (Bogdanov, 2014). Honeydew is a solution

https://en.wikipedia.org/wiki/Sugar


7

with varying sugar concentration (5-60 %), containing mainly sucrose, besides higher

sugars (oligosaccharides). There are also smaller amounts of amino acids, proteins,

minerals, acids and vitamins. Besides, honeydew contains cells of algae and fungi

(Bogdanov, 2014).

1.4. Honey chemical composition

Honey is composed mainly by sugars, glucose and fructose, and in a less amount

water and other components like minerals, vitamins, proteins and amino acids, Table 1.

Table 1. Honey composition after (Bogdanov, 2009) values in g/100g.

Nectar honey g/100g Honeydew honey g/100g

Average Min-Max Average Min-Max

Water content 17.2 15-20 16.3 15-20

Fructose 38.2 30-45 31.8 28-40

Glucose 31.3 24-40 26.1 19-32

Sucrose 0.7 0.1 0.5 0.1-4.7

Other disaccharides 5.0 4.8 4.0 16

Melezitose <0.1 - 4.0 0.3-22.0

Erlose 0.8 - 1.0 0.16

Other

oligosaccharides

3.6 0.56 13.1 0.1-0.6

Total sugars 79.7 0.5-1 80.5 -

Minerals 0.2 0.1-0.5 0.9 0.6-2

Amino acids and

proteins

0.3 0.2-0.4 0.6 0.4-0.7

Organic acids 0.5 0.2-0.8 1.1 0.8-1.5

pH 3.9 3.5-4.5 5.2 4.5-6.5


8

1.4.1. Sugars

Sugars are the main constituents of honey, comprising about 95 % of honey dry

weight (Bogdanov, 2014). The monosaccharides glucose and fructose are the main sugars

found in honey, which are the building blocks of the more complex sugars and are the

resulting products of the disaccharide sucrose hydrolysis (White, 1980). The main

oligosaccharides in nectar honeys are disaccharides: sucrose, maltose, turanose, erlose.

Honeydew honey also contains the trisaccharides melezitose and raffinose. Trace amounts

of tetra and pentasaccharides have also been isolated, including isomaltotetraose and

isomaltopentaose (Bogdanov, 2014).

1.4.2. Water content

Water is the second largest constituent of honey, and its content is also related to

the maturity of this product. The moisture content can be influenced by floral and

geographical origin, climatic factors, season of the year, processing and storage

conditions, as well as the degree of maturity achieved in the hive (Gallina et al., 2010). It

has significant impact on the physical properties of honey, such as, viscosity and

crystallization, but also taste, color, flavor, solubility, conservation and specific gravity

and also in the shelf life of the product. According to the Codex Alimentarius Committee

on Sugars, the moisture content in honey should not exceed 20 g /100 g (Codex

Alimentarius, 2001). If the moisture content is higher, the honey is more likely to ferment

due to the presence of yeasts and osmophilic microorganisms. Since honey is hygroscopic,

the moisture in honey can also increase during the processing operations of the product, as

well as the inadequate storage conditions (White, 1980).

1.4.3. Proteins and amino acids

Proteins and amino acids in honey are originated from both bees (salivary glands),

and plants (nectar, honeydew and mainly pollen). About 20 different non-enzymatic

proteins have been identified in honey (De-Melo et al., 2018). The quantity of proteins can

vary from 0.1 to 0.7%, Table 1. Overheated or long-time stored honeys show a reduction

or absence of protein content (De-Melo et al., 2018). Around 26 amino acids have been

detected in honey, such as proline, glutamic acid, alanine, phenylalanine, tyrosine, leucine,

among others (Cotte and Giroud, 2004). The most abundant amino acid found in honey is

proline, ranging from 50 to 85% of the total. The proline content in honeys should be


9

higher than 200 mg/kg (Bogdanov, 2002). When the values of this amino acid are

significantly lower than 180 mg/kg, the minimum value that has been agreed for genuine

honey, it indicates sugar adulteration. Proline can be seen as quality criteria for honey

ripeness (Von-der, Dustmann, 1991).

1.4.4. Enzymes

The degrees of enzymes present in honey are sometimes used as an indicator for

honey quality, freshness and overheating. Enzymes in honey are originates from the honey

bees or from the plant visited by the bees. Diastase (α- and β-amylase) digests starch to

maltose and is relatively stable to heat and storage and invertase (glucosidase) catalyzes

mainly the conversion of sucrose to glucose and fructose, but also many other sugar

conversions (Raude, 1994). Also, glucose oxidase and catalase regulate the production of

H2O2, one of the honey antibacterial factors (Bogdanov, 2014). The enzyme content also

depends on temperature, honey botanical origin, nectar abundance flow, state and strength

of the colony, seasonal activity of the bee, bee specie, diet, age and physiological stage of

the bee (De-Melo et al., 2018).

Diastase activity is a physicochemical parameter usually investigated as marker of

honey freshness (Fechner et al., 2016; Flores et al., 2015). It can be expressed in Schade,

Göthe or diastase units and honey generally should present a diastase activity of at least 8

Schade units, which is the minimum value accepted by regulatory organizations (Codex

Alimentarius Commission, 2001). Similar to 5-HMF, the diastase activity can be used as an

indicator of aging and increase temperature because it may be reduced during storage or when

the product is subjected to heating above 60 oC (Fechner et al., 2016; Flores et al., 2015).

1.4.5. 5-Hydroxylmethylfurfural (5-HMF)

5-HMF is a product of the decomposition of monosaccharides such as fructose,

Fig. 2. The reaction occurs slowly and naturally during the storage of honey, and much

more quickly when honey is heated. The 5-HMF amount present in honey is the reference

used as a guide to the amount of heating that has taken place; the higher the 5-HMF value,

the lower the quality of the honey (Bear, 2009). However, 5-HMF alone cannot be used to

determine the severity of the heat treatment, because other factors can influence the levels

of 5-HMF, such as the sugar profile, presence of organic acids, pH, moisture content,

water activity and floral source. Therefore, the 5-HMF content gives only an indication of

overheating or inadequate storage conditions (Bogdanov, 2014). As indicated by the


10

Codex Alimentarius and EU standards, the 5-HMF maximum is 40 mg/kg for the mixture

or processed honey, and a maximum of 80 mg/kg for honeys with a tropical origin.

(Bogdanov, 2014).

Figure 2. 5-HMF formation resulting from a sugar decomposition reaction (Bogdanov, 2014)

1.4.6. Organic acids

Honey contains organic acids, in equilibrium with the corresponding lactone,

representing less than 0.5% of total solids. They are important for honey taste, aroma,

color, acidity and honey preservation, making it difficult for microorganisms to grow

(Bogdanov, 2014). Organic acids in honey have different sources, while some acids can

come directly from nectar or honeydew, the majority, are produced from sugars by the

action of enzymes secreted by bees during ripeness and storage (De-Melo et al., 2018).

Gluconic acid is the main honey organic acid, representing the 70–90% of the total

(Bogdanov, 2014). It comes from glucose by the action of glucose oxidase. In addition to

gluconic acid, more than 30 different non-aromatic organic acids were found in honey.

Legally, organic acids should not exceed 50 meq/kg. For honey intended for industry, the

tolerated limit is of 80 milliequivalents (Lequet, 2010).

1.4.7. Vitamins

Honey has small amounts of vitamins, which come mainly from the pollen grains

in suspension (Matzke and Bogdanov, 2003). Vitamins found in honey include thiamine

(B1), riboflavin (B2), nicotinic acid (B3), pantothenic acid (B5), pyridoxine (B6), biotin

(B8), folic acid (B9) and also vitamin C. Those vitamins present in honey are preserved

due to the low pH of honey. The commercial filtration of honey may cause a reduction in

vitamin content due to the almost complete removal of pollen. Also, the loss of vitamins

in honey can happen due to the oxidation of ascorbic acid by the hydrogen peroxide

produced by glucose oxidase (Ciulu et al., 2011).

1.4.8. Mineral content

Mineral composition in honey is generally low, ranging between 0.02 and 0.3% in

nectar honeys, while in honeydew honeys can reach 1% of the total (Felsner et al., 2004).


11

Its content can vary with the soil and climatic conditions, as well as the chemical

composition of the nectars originated from the different botanical sources. Also, the

harvesting and the beekeeping techniques can have influence in the honey mineral

(Felsner et al., 2004) content. The main minerals found in honeys are potassium, sodium,

calcium and magnesium and in lesser amounts iron, copper and, manganese. In minor

quantities, as trace elements, are found boron, phosphorus, sulfur, silicon and nickel,

among others (Doner, 2003). Generally, dark honeys contain more minerals than the light

ones, being higher in honeydew honeys (Bear, 2009). The mineral content is correlated

with the ash percentage and the electrical conductivity (Da Silva et al., 2016).

1.4.9. Volatile compounds

Researchers began the study of honey aromatic substance in the mid of 1960.

Honey volatiles are the substances responsible for the honey fragrance. Most of them are

derived from plants, but also some are included by the honey bees. Until now around 600

compounds have been identified in the volatile fraction of honey, and some are used as

markers of monofloral honeys, such as 3,9-epoxy-1-p-mentadieno, t-8-p-menthan-oxide-

1,2-diol and cis- rose, which have been proposed as markers of lemon honey; diketones,

sulfur compounds and alkanes are characteristic of eucalyptus honey, while hexane and

heptanal are the main compounds in the aroma of lavender honeys (Castro-Vázquez et al.,

2007). Other volatiles from different chemical families are present in honey at very low

concentrations, such as monoterpenes, C13-norisoprenoid, sesquiterpenes, benzene

derivatives and, to a lower content, superior alcohols, esters, fatty acids, ketones, terpenes

and aldehydes (Pontes et al., 2007).

1.4.10. Phenolic compounds

Phenolic compounds are plant-derived secondary metabolites. These compounds

have been used as chemotaxonomic markers in plant systematics. They have been

recommended as potential markers for the determination of botanical origin of honey and

for the differentiation between monofloral and multifloral honeys. In honey, as well as

from pollen or propolis they are mainly derived from plants (Ferreres, Ortiz and Silva,

1992), being present in a range of 5–1300 mg/kg (Gheldof and Engeseth, 2002).

According to the phenolic structural features, polyphenols are divided into two main

groups, phenolic acids and flavonoids (Tomás- Barberan et al., 2001). Flavonoids

aglycones are the mainly polyphenols found in honey. The loss of the sugar moiety of the


12

glycosides present in nectar is due to the hydrolysis by bee saliva enzymes (Tomás-

Barberán et al., 2001). Dark honeys usually contain a higher quantity of phenolic

compounds than the light ones. Dark honeys have been reported to contain more phenolic

acid derivatives but less flavonoids than light ones (Tomás-Barberan et al., 2001).

1.5. Other physicochemical parameters

1.5.1. Color

Honey color can vary from practically colorless to brown dark, sometimes with

green or reddish reflexes. These variations in the color of honey can related to its flavor:

honey with lighter color have a gentle flavor while the darker honeys have a stronger

flavor (Marchini, Sodré and Moreti, 2004). The color of honey depends on its floral

origin, climate factors during nectar flow, soil conditions and the temperature at which the

honey matures in the hive. Also, pollen, sugars, carotenoids, xanthophylls, anthocyanins,

minerals, amino acids and phenolic compounds, mainly flavonoids (Bogdanov et al.,

2004). Furthermore, honeydew honey is darker than bloom honey primarily because of

mineral and phenolic substance and other components (Can et al., 2015).

1.5.2. Electrical conductivity

Electrical conductivity is a property related to the ability of a material to lead an

electric flow. Honey contains minerals and acids, serving as electrolytes, which can

conduct the electrical current, thus, the higher their content, the higher the resulting

conductivity. It is an indicator often used in the quality control of honey that can be used

to distinguish floral honeys from honeydew honeys. At present it is the most useful quality

parameter for the discrimination between floral honeys and honeydew honeys. As this

parameter is directly related to the ash content, it was included in the Codex Alimentarius

Standards, replacing the determination of the ash in honey. The standards recommend a

maximum value of 0.8 mS cm-1

(Codex Alimentarius, 2001; Bogdanov, 2014).

1.5.3. pH and acidity

The pH of honey ranges between 3.5 and 5.5 depending on its floral and

geographical source, the pH of nectar, soil or plant association, and the amount of

different acids and minerals (Crane, 1985). While pH analysis is useful as an auxiliary

variable to estimate the quality of the product and as a parameter for evaluating total

acidity, it is not directly related to free acidity due to the actions of the buffer acids and


13

minerals present in honey (Pereira et al., 2009). The acidity of honey can be assessed as

free, lactonic, and total (free + lactonic) acidity (Navarrete et al., 2005). Free acidity is a

parameter related to the deterioration of honey, being its limit established as 50 meq kg-1

(Codex Alimentarius, 2001; EU Commission, 2002). Higher values may be indicative of

fermentation of sugars into organic acids (Almeida et al., 2013).

1.6. Antibiotic residues in honey

According to Regulation (EC) No 470/2009, no veterinary medicinal product is

permitted in beekeeping products. In fact, no antibiotic has ever had an MRL (Maximum

residue limits) in honey (Cara et al., 2012). However, some countries, like Switzerland,

UK, and Belgium, have established action limits for antibiotics in honey, which generally

lies between 0.01 to 0.05 mg/kg for each antibiotic group (Al-Waili et al., 2012). Some

antibiotics have the potential to produce toxic reactions in consumers directly while some

other can produce allergic or hypersensitivity reactions (Velicer et al., 2004). Antibiotic

residues consumed along with food and honey can produce resistance in bacterial

populations. Antibiotic resistance is a global public health problem and continues to

be a challenging issue (Al-Waili et al., 2012). Two main approaches are used to

determine the content of antibiotic residues in honey: screening tests and multi-stage

analytical methodologies. The simple tests provide qualitative information, enabling

determination of a single target analyte. With multi-stage methods, a fairly broad spectrum

of analytes can be determined in one analytical run. (Barganska, Slebioda and Namiesnik,

2011).

1.7. Biological properties of honey

Honey has been found to contain significant antioxidant compounds including

glucose oxidase, catalase, ascorbic acid, flavonoids, phenolic acids, carotenoid

derivatives, organic acids, amino acids and proteins (Beretta et al., 2005). Research

showed a correlation between color and antioxidant capacity, with the darker honeys

providing the highest levels of antioxidants (Jaganathan and Mandal, 2009).

Phenolic content in honey is responsible for anti-inflammatory effect (Al-Waili,

Boni, 2003). These phenolic and flavonoids compounds cause the suppression of the pro-

inflammatory activities of cyclooxygenase-2 (COX-2) and/or inducible nitric oxide

synthase (iNOS) (Viuda, Ruiz, Fernandez, 2008). Furthermore, ingestion of diluted

natural honey has produced reductions on concentrations of prostaglandins such as PGE2


14

(prostaglandin E2), PGF2α (prostaglandin F2a) and thromboxane B2 in plasma of normal

individuals (Reyes, Segovia and Shibayama, 2007).

1.8. Beekeeping in Algeria

Beekeeping in Algeria is practiced mainly in the north of the country, where the

floral diversity is ensured almost all the year. The honeybees need to be adapted to the

desert climate and to be resistant to unfavorable environmental conditions such as high

temperatures and strong prevailing winds. Hives which are best suited or adapted to the

desert conditions must be used. Traditional hives made from rocks and muds are known

from ancient times in Algerian deserts. Nowadays, Langstroth hive type is used in Algeria,

Fig.3, with modifications due to the hot weather (Moustafa, 2001).

A B

Figure 3. (A) The Langstroth hive and (B) the Langstroth hive different parts (John, 2014).

In 2010, the Algerian Beekeeping Organization, counted around 1.2 million

colonies Fig.4.A, and 20,000 beekeepers. The development of honey production shows a

clear increase from 2002 to 2010, Fig.4.B (Adjlane et al., 2012).

Figure 4. Number of honeybee colonies in Algeria from 2002 to 2010. (B) Honey production

in Algeria from 2002 to 2010. Source: Ministry of Agriculture and Rural Development:

MADR (2009-2010) (Adjlane, Doumandji and Haddad N. al., 2012).

0

5

10

15

2002 2004 2006 2008 2010

colo

nie

s num

ber

x 1

00

00

0

years

0

1

2

3

4

5

2002 2004 2006 2008 2010

ho

ney

pro

duct

ion (

on

mil

lio

n o

f kg)

years


15

A B

In Algeria, there are two main bee subspecies. The Tellian bee (Apis mellifera

intermissa), Fig.5-(A), is native of the region located between the atlas and the

Mediterranean which is known by the name of Tell. It is characterized by its black

abdomen and its agressivity. The main advantages of this bee are its longevity, remarkable

ability to harvest pollen and a high production of honey which can reach up to 100 kg per

colony provided that modern beekeeping methods are applied (Fresnay, 1981).

The Saharan (desert) bee (Apis mellifera sahariensis), Fig.-5(B), better known as

the Sahara bee, or locally the yellow bee. It is recognized for its many advantageous

features such as the high breeding, the precocity, the extraordinary aptitude for nectar and

pollen harvesting and good adaptability under difficult climatic conditions (Kessi, 2013).

Figure 5. Images showing (A): Apis mellifera intermissa bee and a (B): Apis mellifera

Sahariensis bee (Tlemcani, 2013).

1.9. Algerian honey

In this research, representative Algerian honeys such as, Euphorbia (Euphorbia

bupleuroides), jujube (Ziziphus lotus), Eucalyptus (Eucalyptus globulus) and multifloral

honeys will be focused.

1.9.1. Eucalyptus honey

The eucalyptus tree is a large, fast-growing evergreen that is native

from Australia and Tasmania. The tree can grow to 125-160 meters. Eucalyptus belongs to

the Myrtaceae family and more than 300 species of eucalyptus are described as

Eucalyptus globulus, Fig 6.A, which is the most common and well-known (Catherin,

2020). Many of which produce enough nectar for honey bees to yield appreciable amounts

of honey (Catherin, 2020; Persano, Baldi and Piazza, 2004). The main physicochemical

parameters are shown in, Table2. It is a honey with a clear amber color, a wet wood, very

intense and persistent aroma, a sweet with a slight acid note and a medium tendency for

crystallization with fine crystals (Orantes et al., 2018).

https://www.encyclopedia.com/places/australia-and-oceania/australian-and-new-zealand-political-geography/australia

https://www.encyclopedia.com/places/australia-and-oceania/australian-and-new-zealand-political-geography/tasmania


16

1.9.2. Euphorbia honey

Euphorbia is one of the largest flowering plant in the spurge family

(Euphorbiaceae). With over 2,000 species, euphorbias can range from tiny annual plants

to large and long-lived trees and look completely different. In the deserts of Africa and

Madagascar, euphorbia adapted its physical characteristics becoming similar to cacti of

America, although they are not cacti (Cherif et al., 2011). Recent inventory of native

plants in Algeria identify over 51 species of Euphorbiaceae, where E. bupleuroides,

Fig.6.B, is the main species used by bees to produce honey (Le Houèrou, 1995; Quezel

and Médail, 2003).

Table 2. Physicochemical properties of Jujube, Euphorbia, Eucalyptus honeys of arid and

semi-arid zones in north Africa (Cherif et al., 2016; Cherif et al., 2016); (Makhloufi et al.,

2010)

The main physicochemical parameters are shown in Table 2. It is a honey with

golden yellow to dark amber color, with a sweet, pinch in the throat with a typical light bit

back flavor and with a spicy almost peppered aroma and pungent flavor (Cherif et al.,

2011).

Botanical

origin

pH Electrical

conductivity

s/cm

Water

content

%

Diastase

Schade

unit

Sucrose

%

5- HMF

mg/kg

References

Ziziphus 4.4 673 16.65 15.63 0.61 8.71 (Cherif et al.,

2016)

Euphorbia 4.2 411 17.06 12.67 0.97 12.08 (Cherif et

al., 2011)

Eucalyptus 4.2 769 16.5 9.64 25.63 (Makhloufi

et

al., 2010)


17

A B C

Figure 6. (A) Eucalyptus plant (Orantes, Gonell, Torres et al., 2018). (B) Euphorbia plant. (C)

Jujube plant (Photograph by Andrii Salomatin,

https://www.shutterstock.com/fr/g/Andrii%2BSalomatin retrieved on 24-05-2

1.9.3. Jujube honey

Ziziphus lotus L. belongs to the family Rhamnaceae, which consist of about 135

species. The trees are medium-sized, growing 7-10 meters high, with shiny green leaves

about 5 cm long. The edible fruits are a globose dark yellow drupe with 1–1.5 cm

diameter, Fig.6.C. The wild jujube Ziziphus lotus is a species found in many habitats of

arid and semiarid regions of the Mediterranean area, throughout Libya to Morocco and

Algeria (Benammar et al., 2010).

Jujube honey is a highly demanded product in Algeria and worldwide, being

considered one of the most expensive honeys. Despite the commercial interest, this honey

type has been scarcely described (Cherif et al., 2016). The main physicochemical

parameters of jujube honey are shown in Table2. Its color is varied from light-amber to

amber.

https://en.wikipedia.org/wiki/Fruit

https://en.wikipedia.org/wiki/Drupe

Chapter II- Materials and methods

18



19

2.Material and methods

2.1. Honey samples This work was carried out with ten Algerian monofloral and multifloral honey samples,

obtained from local beekeepers and harvested in 2019, Fig.7. The honey samples were

stored in the original containers at room temperature.

Figure 7. Geographic origin of the honey samples.

In Table 3, there is information regarding the honey samples used throughout this

work, namely their geographical origin, year of production and other relevant information on

the label. Also the probable floral origin, given by the label, is shown in the Table 3.


20

Table 3. Geographic origin and other information from honey samples.

Samples Floral origin on the

label

Geographic origin Collection

year

EC1 Eucalyptus Sidi Belabes 2019

EC2 Eucalyptus Sidi Belabes 2019

MF1 Multifloral Sidi Belabes 2019

MF2 Multifloral Sidi Belabes 2019

J1 Jujube Ein Safra 2019



EF1 Euphorbia El bayed 2019



2.2. Honey analysis

The honey characterization was carried out through the identification of their floral

origin by pollen analysis and by the evaluation of the physicochemical

parameters, defined by the International Honey Commission (IHC) (International Honey

Commission. 2009). Also, the composition of proteins, phenolic compounds and

antioxidant activity was evaluated. All parameters were evaluated in triplicate.

2.2.1. Pollen analysis

For pollen analysis, 10 g of honey, for each sample, were dissolved in 20 mL of

distilled water and centrifuged at 3500 rpm for 10 min. After discarding the supernatant

liquid, 2 mL of glacial acetic acid were added and vortexed. The tube was centrifuged in

the same conditions and the supernatant discarded. Then, 2 mL of the acetolysis solution


21

(acetic anhydride: sulphuric acid, 9:1) were added and the solution vortexed. The tube was

placed in a boiling water bath for 3 min. After cooling and centrifuged, the supernatant

was discarded and 4 mL of 50% glycerol solution was added followed by another step of

centrifugation and removal of the supernatant. A volume of liquefied glycerol-gelatin was

added and immediately vortexed. Then, 17 µL of the mixture were pipetted and spread on

a slide at 40 oC. The slides were allowed to rest, at room temperature, in an invert

position. After sealing the coverslips with nail varnish, the slides were observed under an

optical microscope, at 1000X magnification, 500-1000 pollen grains per sample and

complete lines were counted and identified at random in the coverslip area (Von Der et al,

2004). This work was done in collaboration with LabApisUTAD

.

2.2.2. Physicochemical analysis

2.2.2.1. Color

The color intensity of honey samples was measured according to the Pfund scale.

Briefly, homogeneous honey samples were transferred into a cuvette with a 10 mm light

path until the cuvette was approximately full. Then, the cuvette was inserted into a C221

colorimeter (Hanna Instruments, Woonsocket, RI, USA). color grades were expressed in

millimeter (mm) Pfund grades, compared to an analytical-grade glycerol standard.

2.2.2.2. Moisture content

Moisture content was determined using a hand refractometer (Digit-5890, Ref:

8100.5890), expressing the results in percentages.

2.2.2.3. Electrical conductivity

A honey solution was prepared by diluting 20 g of anhydrous honey in 25mL of

deionized water, and the respective electrical conductivity was measured with the help of a

calibrated Consort C868 conductivity meter (Hanna Instruments, Woonsocket, RI, USA),

Fig. 8. The results are expressed in mS.cm-1

.

Figure 8. Conductivity meter.


22

2.2.2.4 pH, free and lactonic acidity

Free acidity, pH, lactone acidity and total acidity measurements were performed

according to IHC (the International Honey Commission (Bogdanov, 2002). Briefly, 5 g of

honey were dissolved in 25 mL of deionized water, which were pipetted into a beaker where

the pH electrode was immersed and the initial pH value was read. This solution was titrated

with 0.119 M sodium hydroxide, NaOH. The volume spent to reach the equivalence point

(pH=7) was recorded, and the obtained value allowed the determination of the free acidity.

Immediately, an additional volume of 0.119 M NaOH to complete 10 mL was added, and

without delay, back-titrated with 0.022 M sulfuric acid, H2SO4, to pH 7, and so obtaining the

lactonic acidity. Total acidity results were obtained by adding free and lactone acidities. The

results are expressed in meq.kg-1

of honey. The titrations were done using a HI902

potentiometer titrator (Hanna instruments, pH 211 microprocessor pH meters), Fig. 9.

Figure 9. Potenciometer titrator.

2.2.2.5. Proline

The proline content in honey samples was measured weighting 0.5 g of honey into a

volumetric flask and dissolved in about 10 mL deionize distilled water. Then, 0.5 mL of

diluted honey solution was placed in a test tube, 0.5 mL of deionized water (blank test) into

a second tube, and 0.5 mL of proline standard (0.032 M) solution into a third tube. After, 0.5

mL of deionized water, 1 mL of formic acid (98%) and 1 mL of ninhydrin solution (3%)

were added to each tube. The tubes were capped carefully and shaken vigorously. After,

they were placed in ultrasound for 15 min followed into a water bath at 100°C for 15 min

and then transferred to a water bath at 70°C for 10 min. Finally, 5 mL of 2-propanol (50%)

was added and the tubes were capped immediately. After the tubes were allowed to cool

down for 45 min, the absorbance was measured at 510 nm using a UV/Vis

spectrophotometer (Specord 200 spectrophotometer, Analytikjena, Jena, Germany). Proline

content of honey, in mg/kg, was calculated according to following equation:


23

Equation 1. Proline= ((Abs sample)/(Abs standard)) × ((Weight standard)/(Weight sample)) ×80

2.2.2.6 5-Hydroxymethylfurfural (5-HMF)

For the 5-HMF quantification, 5 g of honey were weighted and dissolved in 25 mL

of deionized water and transferred quantitatively into a 50 mL volumetric flask. Then, 0.5

mL Carrez solution I and Carrez solution II were added, completing the final volume of 50

mL with deionized water. The solution was filtered through Waltman paper, rejecting the

first 10 mL of filtrate. The filtrate was pipetted into each of two test tubes. To one of the

tubes, 5 mL of distilled water (sample solution) was added and to the other 5 mL of sodium

bisulphite solution, NaHSO3, 0.2% (reference solution). The absorbance was measured at

284 nm and 336 nm in a spectrophotometer (Specord 200 spectrophotometer, Analytikjena,

Jena, Germany), and the 5-HMF value was expressed in mg/kg and determined according to

the following equation:

Equation 2. HMF= (Abs284-Abs336) ×149.7× (5/ (sample weight))

2.2.2.7. Diastase activity

For the measurement of the diastase index the Phadebas method (Bogdanov, 2002)

was used. For that, 0.1g of honey was weighed, quantitatively transferred to a 10 mL

volumetric flask and completed the volume with 0.1M acetate buffer (pH=5.2). After

preparing the solution, 5 mL were added to a test tube (sample) and placed in a water bath of

40 °C, together with a second tube (reference solution) containing instead 5 mL of 0.1 M

acetate buffer solution (pH 5.2). Then, the Phadebas tablets were added to the two tubes,

which, after mixing, were kept at 40ºC for 15 minutes, After this time, The absorbance was

measured at 620 nm using a spectrophotometer (Specord 200 spectrophotometer,

Analytikjena, Jena, Germany). The result was presented as diastase index (DN), in Schade

units, corresponding to a unit of diastase and the enzymatic activity of 1 g of honey capable

of hydrolyzing 0.01 g of starch at 40ºC in one hour. The formulas used to calculate the DN

value were as follows:

Equation 4. DN= 28.2*Abs620 + 2.64, if DN > 8

Equation 3. DN= 35.2*Abs620 – 0.46 if DN<8


24

2.2.3. Sugar analysis

For sugars analysis, about 2.5 g of honey was mixed with 20 mL of deionized water

and 12.5 mL of methanol and 1 mL of xylose (internal standard, 30mg/mL) and the resulting

solution was diluted to a final volume of 50 mL with deionized water. Afterwards, the

sample was passed through a 0.2 μm filter and analyzed by high performance liquid

chromatography coupled to a refractive index detector (HPLC-RI). HPLC-RI was performed

on an integrated Knauer system with pump (Smartline 1000), a degasser (Smartline 5000), a

UV detector (Knauer Smartline 2300) and an autosampler (Jasco, AS-2057). Data

acquisition and remote control of the HPLC system was done by Clarity Chrom software

(Knauer, Berlin, Germany). The chromatographic separation was achieved using a

Eurospher 100-5 NH2 (4.6 × 250 mm, 5 mm, Knauer) column at 30 ˚C. The mobile phase

was composed by acetonitrile/water, 80:20 (v/v) at a flow rate of 1.3 mL/min. The

identification of sugars was obtained by comparison of retention time between samples and

standards. Quantification was achieved using calibration curves of Table 4.

Table 4. Calibration curve for sugars.

Sugars Calibration curve R2

Fructose y = 82.665x + 75.806 0.9900

Glucose y = 60.65x + 154.24 0.9903

Sucrose y = 66.558x + 58.629 0.9907

Trehalose y = 86.976x + 0.7149 0.9900

Turanose y = 129.76x - 10.213 0.9983

Maltulose y = 71.156x + 1.4642 0.9976

Maltose y = 65.454x - 2.224 0.9996

Melezitose y = 58.269x + 18.123 0.9903

Raffinose y =53.431x + 12.721 0.9941

Melibiose y = 32.126x +6.8297 0.9903

Kojibiose y= 95.399x + 1.8282 0.9981

Erlose y = 60.749x + 9.616 0.9913

Isomaltose y = 57.638x - 1.958 0.9968


25

2.2.4. Minerals

For the test of the minerals content, the following elements were assessed: magnesium

(Mg), calcium (Ca), sodium (Na), and potassium (K), via the spectrophotometer of flame

atomic absorption: Pye Unicam PU9100X. The detection of manganese (Mn), copper (Cu)

cadmium (Cd) and lead (Pb) was done using atomic absorption spectrophotometry thought

graphite chamber via a Perkin Elmer PinAAcle 900 spectrophotometer.

2.2.4.1. Sample Digestion

A sample of 1g was weighted into a PTFE digestion tube then 10 mL of concentrated

nitric acid (HNO3) was added. The sample was digested in a microwave via the following

temperature gradient sequencer: a power of 1200 W during 15 minutes until 200ºC. The

continuous of these conditions were sustained for another 15 minutes. After that, it was

cooled and quantitatively transferred into a volumetric flask of 50 mL.

2.2.4.2. Sample Analysis

The quantification of the different minerals required a previous preparation for

specific solutions and standards according to the following procedures:

2.2.4.2.1. Potassium and Sodium

For the quantification of the sodium and potassium elements, a cesium chloride

buffer (10 g/L) and the preparation of different standard solutions were done according to

the following requirement: solution 1: 10 mL of the potassium standard (1000 ppm) and 5

mL of sodium standard (1000 ppm) were pipetted into a flask of 20 mL and the volume

completed with deionized water. Then the dilution of this stock solution was done further,

according to (Table 5), for presenting the calibration standards as follows.


potassium and sodium.

Standard V(sample)/mL Vf/mL

P1/4 0.25

50

P1/2 0.25

P1 1.00

P2 2.00

P3 3.00

P4 4.00

P5 5.00


26

The calibration standards were done in the spectrophotometer resulted from the ten-

fold dilution of these standards (5.0 mL solution of each standard and 5 mL CsCl buffer in a

final volume of 50 mL). For the analysis of potassium, a digested supplement solution of 5

mL, buffer solution of 1 mL and 4 mL of deionized water were added. For the analysis of

sodium, 10 mL of the digested supplement solution, 1 mL of the buffer solution were added.

The recording of the result was taken according to the conditions suggested for the tools.

2.2.4.2.2. Calcium and Magnesium

For the detection and quantification of calcium and magnesium, a solution (10 g/L)

of lanthanum was prepared by diluting 13.15 g of La(NO3)2 in 1L of deionized water. Also,

a Ca standard solution (1000 ppm, solution 2) and an Mg standard solution (1000 ppm,

solution 3) was set in 10 ml of deionized water. Also, from stock solutions 2 and 3 a series

of standard solutions were set according to the following (Table 6).


calcium and magnesium.

Standard V (sol 2)/mL V (sol 3)/mL Vf/mL

P1/4 0.25 0.25 50

P1/2 0.25 0.25

P1 1.00 1.00

P2 2.00 2.00

P3 3.00 3.00

P4 4.00 4.00

P5 5.00 5.00

The standards applied in the spectrophotometer calibration to determine the content

of Ca are done from the ten-fold dilution of these standards (5.0 mL solution of each

standard and 5 mL of solution La to a final volume of 50 mL). The standards applied in the

spectrophotometer calibration to determine the content of Mg were done from the thirty-

three-fold dilution of these standards (1.50mL solution of each standard and 5 mL of

solution La to a final volume of 50mL). To detect the content of potassium in the

supplement, a digested supplement solution of 5 mL, buffer solution of 1 mL and 4 mL of

deionized water were added. For the quantification, a digested solution of 10 mL and

lanthanum solution of 1 ml was added. To determine the Ca and Mg the recommended

condition according to the equipment was followed.


27

2.2.4.2.3. Iron

Matrix modifier: diluted 1.7mL of magnesium nitrate solution, Mg(NO3)2, 10 g/L to

10 mL of solution with deionized water.

Standard 1: diluted 0.50 mL of 1000 ppm standard solution to 50mL with deionized

water.

Standard 2: diluted 0.50 mL of standard solution to 50 mL with deionized water.

The standards used to construct the calibration curve resulted from the automatic

dilution of standard 2 according to the table. For sample analysis, 20 µL of the sample was

pipetted from a 5 µL matrix modifier. The instrumental conditions recommended for iron

analysis were used.

Table 7. The calibration standards used in the spectrophotometer for the determination of iron.

Standard V(P2) /µL V(Matrix)/µL V (H2O) /µL

P1/4 5 5 15

P1/2 10 5 10

P3/4 15 5 5

P1 20 5 0

2.2.4.2.4. Lead

Matrix modifier: 0.10 mL of magnesium nitrate solution, Mg(NO3)2, and 1.0 mL of

10% monobasic ammonium phosphate solution were diluted to 10mL of solution with

deionized water.

Standard 1: 0.50 mL of 1000 ppm standard solution was diluted to 50 mL with

deionized water.

Standard 2: 0.70 mL of standard 1 solution was diluted to 50 mL with deionized

water.

The standards used to construct the calibration curve resulted from the automatic

dilution of standard 2, according to Table 8.

For the sample analysis, 20µL of the sample was pipetted with a 5 µL of matrix

modifier. The instrumental conditions for the analysis of lead were used.


28

Table 8. The calibration standards used in the spectrophotometer for the determination of lead

Standard V(P2) /µL V(Matrix )/µ

L

V (H2O) /µL

P1/4 5 5 15

P1/2 10 5 10

P3/4 15 5 5

P1 20 5 0

2.2.4.2.5. Manganese, Copper, and Cadmium

To determine the content of manganese, a modified matrix was applied by the

dilution of 1.7 mL of a magnesium nitrate solution, Mg(NO3)2, 10 g/L to final volume of 10

mL with deionized water. Two standards for manganese were done, one diluting 0.50 mL of

standard solution (1000 ppm) to a final volume of 50 mL of deionized water and another by

the dilution of 0.20 mL of the previous solution to a final volume of 50 mL of deionized

water (standard 2). For copper, a modified matrix resulted from the dilution of 1.0 mL of

palladium solution, Pd, 10 g/L, and 0.1mL of magnesium nitrate solution, Mg(NO3)2, to a

final volume of 10 mL of solution in deionized water. After that, the preparation of two

copper standards was done by the dilution of 0.50 mL of the 1000 ppm standard solution (Vf

= 50 mL deionized water, standard 1) and the dilution of 0.50mL of the previous solution to

a final volume of 50mL (standard 2). To determine the cadmium content, preparation of

modified matrix was done by the dilution of 0.10 mL of magnesium nitrate solution,

Mg(NO3)2, and 1.0 mL of 10% monobasic ammonium phosphate solution, NH4H2PO4, in 10

mL of deionized water. The preparation of two standard solutions was then done, the first by

the dilution of 0.25 mL of standard solution (1000 ppm) to 50 mL with deionized water

(standard 1) and the second, by dilution of 0.10 mL of the above solution to 50 mL with

deionized water (standard 2). The standards applied for the construction of the calibration

curve resulted from diluting standard 2, according to (Table 9). To analyze all the samples,

20 μL of sample and 5 μL of the modified matrix were pipetted with the application of the

recommended instrumental conditions for each one of the analyses.


manganese, copper, and cadmium.

Standard V(P2)/mL V(matrix)/mL V (H2O)/µL

P1/4 5 5 15

P1/2 10 5 10

P1 15 5 5

P2 20 5 0


29

2.2.3. Nutritional parameters

2.2.3. Ash content

The ash content was determined, in triplicate, indirectly through its calculation, according

to the defined in the literature (Sancho el al, 1992) using the following formula:

Equation 6. % Ash= (conductivity/1000)-0.14/1.74

2.2.3.2. Protein content

For the determination of the protein content,1 g of honey sample was weighed into a

250 mL test tube, 2 catalyst tablets (9% CuSO4) and 15 mL concentrated sulphuric acid

(98%) were added. The blank was prepared with all chemicals and without sample; 5mL of

distilled water was used instead of sample. Samples were digested for 70 minutes at 400 °C.

Before distillation and titration, the test tubes were let to cool down to 50-60 °C, then 25 mL

of distilled water was added to the mixture. The samples were distilled according to the

following parameters; HCl (0.2M) as titrant solution, NaOH (32 %): 50 mL, H3BO3 (4 %

with indicators): 30 mL. For the conversion of nitrogen content into total protein, a factor of

6.25 was used, expressing the results in g/100 g of honey.

2.2.3.3. Total Carbohydrates:

The carbohydrate content of the honey samples was obtained by differential

calculation considering the following expression defined in the literature (Nogueira et al,

2012):

Equation 7. % Total carbohydrates = (100% -Moisture)- (% ash+%protein+%lipids)

2.2.3.4. Energy

The energy value expressed in kcal was calculated in 100g of honey, using the

following equation (Estevinho et al, 2012):

Equation 8. Energy value (kcal/100g) =4× (%protein+%carbohydrates) +9× (%lipid)


30

2.3. Spectrophotometric analysis of the phenolic compounds

2.3.1. Total phenolic content

For the total phenolic content, 1 g of honey sample was diluted with 10 mL methanol.

Then, an aliquot of 0.5 mL of the solution was mixed with 0.5 mL of the Folin–Ciocalteu

reagent and 1 mL of a 20% sodium carbonate solution. Deionized water was added to a final

volume of 5 mL. Following the incubation of 1 hour, the absorbance of the reaction mixture

was measured at 760 nm using a spectrophotometer (Analytik Jena, Jena, Germany). Gallic

acid was used (0.005–0.15 mg/mL) as the standard solution and the values expresses as

milligram of gallic acid equivalent per g of sample (mg GAE/g).

2.3.2Total flavonoid content

Total flavonoid content was determined using the aluminum chloride (AlCl3)

colorimetric method (Alothman, Bhat and Karim, 2009). The Al3+

cations form stable

complexes with free hydroxyl groups of flavonoids this causes the extension of the

conjugated system a shift of the absorption maxima to a longer wavelength region, allowing

quantification in a spectrophotometer at 415 nm (Buriol et al, 2009). The honey solutions

were prepared at the concentration of 0.1 g/mL. One milliliter of the stock solution was

diluted with 10 mL of methanol and then mixed with 0.5 mL of a 5% aluminum chloride

solution (2% aluminum chloride in 5% acetic acid/methanol) and the volume adjusted to 5

mL with 5% acetic acid/methanol. Following incubation for 30 min, in the dark at room

temperature, the absorbance was measured at 415 nm using a spectrophotometer (Analytik

Jena, Jena, Germany). Quercetin was used to calculate the standard curve (0.0016-0.5

mg/mL) and the results were expressed as mg of quercetin equivalents per g of sample (mg

QE/g).

2.4. Phenolic compounds

2.4.1. Extraction

Extraction of polyphenols from honey is generally accomplished using either liquid–

liquid extraction (LLE) or solid-phase extraction (SPE). In both methods, the first step is to

separate the sugars, which make up the great majority of the honey mass. In our case SPE

followed by LLE were used. For that, 25 g honeys were mixed with 125 mL of acidified

water (pH 2 with HCl) until completely fluid and filtered through cotton to remove solid

particles. The extraction was conducted in a glass column (25 cm x 2 cm) fitted with an


31

opening valve and a fritted glass support. The column was packed with 25 g of

Amberlite®XAD

®-2 in methanol, Figure 10. The phenolic compounds remained in the

column, while sugars and other polar compounds eluted with the water. After passing the

honey solution, the column was washed with the acidified water and then with deionized

water. Then, the phenolic fraction was eluted with methanol and the solution evaporated

under reduced pressure at 40 oC. The residue was re-dissolved in 5 mL of water and

extracted with diethyl ether (5 mL x 3). The resulting extracts were combined, concentrated

under reduced pressure and re-dissolved in methanol for further LC-MS analysis.

Figure 10. Phenolic compounds extraction stages; acidified water (pH 2) (A), deionized water

(B), and methanol (C).

2.4.2. Phenolic profile by UPLC / DAD / ESI-MSn

The phenolic compounds characterization was made through UPLC / DAD / ESI-

MSn performed on a Dionex UPLC 3000 equipment (Thermo Scientific, USA) (Figure 11)

equipped with a photodiode detector and coupled to a mass detector. The chromatographic

system consisted of a quaternary pump, an automatic sampler maintained at 5ºC, a degasser,

a photodiode array detector and an automatic thermostatic column compartment. The

2 cm

1-Column packing: 25g resin

2- Conditioning: 50 mL A

3- Sample loading: 25g of honey solubilized

in 125 mL of A

4-washing: 50 mL of A then 150 mL of B

5- Elution:150 mL of C

Purification


32

chromatographic separation was performed on a U-VDSpher PUR C18-E 100 mm x 2.0 mm

i.d. column, with particle size of 1.8 μm (VDS Optilab, Germany), maintained at 30ºC. The

mobile phase was composed of (A) 0.1% (v / v) formic acid in water and (B) 0.1% (v / v)

formic acid in acetonitrile, previously degassed and filtered using a nylon membrane with

0.22 μm porosity. A linear gradient with a flow rate of 0.3 mL/min was used: 0.0-1.0 min 20%

B ; 1.0-11.1 min 20-95% (B); 95% (B) for 2 min; 13.1-13.3 min 95-20% (B); and 20% (B)

for 5 min. The injection volume was 3 μl. Spectral data for all peaks were detected in the

range 190-600 nm. Each sample was filtered through a 0.2 µm nylon membrane (Whatman).

Mass analysis was performed on a LTQ XL mass spectrometer (Thermo Scientific, CA,

USA), in negative mode, equipped with an ESI electro spray ionization source: spray

voltage, 5 kV; capillary voltage, -20V; capillary tube voltage, -65V; capillary temperature,

325 ° C; gas flow and auxiliary gas (N2), 50 and 10 (arbitrary units), respectively. Mass

spectra were acquired in the mass range 100-1000 m/z. Mass spectra were acquired by full

range acquisition covering 100–1000 m/z. For the fragmentation study, a data dependent

scan was performed by deploying collision-induced dissociation (CID). The normalized

collision energy of CID cell was set at 35 (arbitrary units). Data acquisition was performed

using the Xcalibur® software (Thermo Scientific, CA, USA). Quantification was performed

with standard substance calibration curves for p-hydroxybenzoic acid (y = 4x106x-134082;

R2 = 0.9988), caffeic acid (y = 3x10

6x-12895; R

2 = 0.9998), p-coumaric acid (y = 4x10

6x-

13435; R2 = 0,9999), quercetin (y = 893859 x-11231; R

2 = 0.9999), chrysin (y = 5x10

6 x-

32533; R2 = 0.9990), naringenin (y = 5x10

6 + 14548, R

2 = 0.9996) and abscisic acid (y =

2x107x-4x10

6; R

2 = 0.9988). When standards were not available, the compounds were

expressed by equivalents of the structurally more similar phenolic compound. The

elucidation of the structure of phenolic compounds was carried out by comparing their

chromatographic behavior, UV spectra and mass profile with that obtained for commercial

standards and also with the information obtained in the literature when these were not

available.


33

Figure 11. UPLC / DAD / ESI-MSn

equipment

2.5. Antioxidant activity

2.5.1. DPPH˙ assay

The antiradical activity of the honey samples was estimated using the 2, 2-diphenyl-

1-picrylhydrazyl hydrate radical (DPPH˙). For that, 1g of honey was dissolved in 10 mL of

methanol 20 %. Using a microplate, sample solution, methanol and DPPH were added as

described in the Table10. The absorbance was read at 515 nm using an ELX800 Microplate

Reader (Bio-Tek Instruments, Inc.). Different sample concentrations were used in order to

obtain antiradical curves for calculating the EC50 values, according to the following equation:

%Inhibition = [(Abs DPPH−Abs sample)/Abs DPPH.] × 100

For comparison a standard solution of gallic acid was used with an average value of

EC50 of 1.22 mg/mL.

Table 10. DPPH assay steps.

Well Volume (L)

A 10 L Sample solution +140 L methanol+150 L DPPH

*3

B 20 L Sample solution+130 L methanol+150 L DPPH

*3

C 40 L Sample solution +110 L methanol+150 L DPPH

*3

D 60 L Sample solution + 90 L methanol+150ul DPPH *3

E 80 L Sample solution +70 L methanol+150 L DPPH

*3

F 100 L Sample solution +50 L methanol+150 L DPPH

*3

G Blanc (150 L methanol+150 L DPPH) *3


34

2.5.2. Reducing power activity

The reducing power of honey samples was measured by the ferricyanide prussian

blue assay. Through this assay the capacity to convert Fe3+

into Fe2+

is determined,

measuring the absorbance at 700 nm (Ferreira et al., 2009). A volume of 0.125 mL of honey

sample (0.1g/mL) was mixed with 1.125 mL of phosphate buffer (0.2 mol/L, pH 6.6) and

1.250 mL of 1% potassium ferricyanide. The mixture was incubated in a water bath at 50 ºC

for 20 min at 100 rpm. Then, 1.250 mL of 10% trichloroacetic acid was added to the mixture

and centrifuged at 3000 rpm (Centurion K2R series) for 10 min. The supernatant (1.250 mL)

was mixed with deionized water (1.250 mL) and FeCl3 (0.250 mL, 0.1%), and the

absorbance was measured at 700 nm. Gallic acid was used as standard (0.001-0.01 mg/mL),

and results were expressed as milligram of gallic acid equivalent per 100 g dry of sample

(mg GAE/100 g).

2.6. Cytotoxic potential

The following human tumor cell lines were used: AGS (gastric adenocarcinoma),

CaCo (colorectal adenocarcinoma), MCF-7 (breast adenocarcinoma), and NCI-H460 (lung

carcinoma). A non-tumor cell line, Vero (African green monkey kidney), was also tested.

All of them were maintained in RPMI-1640 medium supplemented with 10% fetal bovine

serum, glutamine (2 mM), penicillin (100 U/mL) and streptomycin (100 mg/mL), with the

exception of Vero, that wasmaintained in DMEM medium supplemented with fetal bovine

serum (10%), glutamine and antibiotics. The culture flasks were incubated in an incubator at

37ºC and with 5% CO2, under a humid atmosphere. The cells were used only when they had

70 to 80% confluence. A known mass of each of the extracts (8 mg) was dissolved in H2O (1

mL), in order to obtain the stock solutions with a concentration of 8 mg/mL. From which

successive dilutions were made, obtaining the concentrations to be tested (0.125 - 8 mg/mL).

Each of the extract concentrations (10 μL) were incubated with the cell suspension (190 μL)

of the cell lines tested in 96-well microplates for 72 hours. The microplates were incubated

at 37ºC and with 5% CO2, in a humid atmosphere, after checking the adherence of the cells.

All cell lines are tested at a concentration of 10,000 cells/well, except for Vero in which a

density of 19,000 cells/well was used. After the incubation period, the cells were corrected:

TCA (10% w/v; 100 μL) was previously cooled and plates were incubated for 1 hour at 4ºC,

washed with water and, after drying, a SRB solution (0.057%, m/v; 100 μL) was added, left

to stand at room temperature for 30 minutes. To remove non-adhered SRB, plates were

washed three times with a solution of acetic acid (1% v/v) and placed to dry. Finally, an


35

adhered SRB was solubilized with Tris (10 mM, 200 μL) and the absorbance at a

wavelength of 540 nm was read in the Biotek ELX800 microplate reader. The results are

expressed in terms of the concentration of extract with the ability to inhibit cell growth by

50% - GI50. As a positive control ellipticin was used.

2.7. Anti-inflammatory activity

The extracts were dissolved in H2O in order to obtain a final concentration of 8

mg/mL. From which successive dilutions were carried out, obtaining the concentrations to

be tested (0.125 - 8 mg/mL). The RAW 264.7 mouse macrophage cell line, obtained from

DMSMZ - Leibniz - Institut DSMZ - Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH, was grown in DMEM medium, supplemented with heat-inactivated

(SFB) fetal serum (10%), glutamine and antibiotics, and kept in an incubator at 37ºC, with

5% CO2 and under a humid atmosphere. Cells were detached with a cell scraper. An aliquot

of the cell suspension of macrophages (300 μL) with a cell density of 5 x 105 cells/mL and

with a proportion of dead cells below 5% according to the Trypan blue exclusion test, was

placed in each well. The microplate was incubated for 24 hours in the incubator with the

conditions previously indicated in order to allow an adequate adherence and multiplication

of the cells. After that period, the cells were treated with different concentrations of extract

(15 μL, 0.125 - 8 mg/mL) and incubated for one hour, with the range of concentrations

tested being 6.25 - 400 μg/mL. Stimulation was performed with the addition of 30 μL of the

liposaccharide solution - LPS (1 mL/mL) and incubated for an additional 24 hours.

Dexamethasone (50 mM) was used as a positive control and samples in the absence of LPS

were used as a negative control. Quantification of nitric oxide was performed using a Griess

reagent system kit (nitrophenamide, ethylenediamine and nitrite solutions) and through the

nitrite calibration curve (100 mM sodium nitrite at 1.6 mM) prepared in a 96-well plate. The

nitric oxide produced was determined by reading absorbances at 540 nm (ELX800 Biotek

microplate reader, Bio-Tek Instruments, Inc., Winooski, VT, USA) and by comparison with

the standard calibration line. The results were calculated through the graphical

representation of the percentage of inhibition of nitric oxide production versus the sample

concentration and expressed in relation to the concentration of each of the extracts that

causes the 50% inhibition of nitric oxide production - IC50.

2.8. Detection of antibiotics residues


36

The Charm II test uses an antibody (as a binder) with specific receptor sites that bind all

of the target antibiotics. The binder is added to a sample extract followed by addition of an

exact amount of H3 or C

14 labeled antibiotics (as a tracer). Firstly, the unknown antibiotics in

the sample combines with the receptor sites and then the radio labeled antibiotics occupy the

remaining sites. After this reaction is complete, a scintillation fluid is added and the

concentration of either H3 or C

14 associated with the binder is measured in counts per minute

(CPM) using the Charm II system (Charm LSC 7600, Charm Science Inc., USA), Figure 12.

Samples with high counts are considered negative (tracer antibiotics are largely bound to the

binder) and samples with low counts are considered positive (tracer antibiotics are largely

free in solution). Thus, the greater the counts, the lower the original antibiotic concentration

in the samples (Kwon et al, 2011).

Figure 12. Charm LSC 7600

The detection of tetracycline and sulphonamide followed the operator’ s manuals

attached to the device.

2.8.1.Tetracycline residues

The charm II tetracycline test for honey is a rapid immunoreceptor assay for the

detection of tetracyclines in honey at 10 to 20 ng/g or parts per billion (ppb). For that, 5 g of

sample were weighted into a centrifuge tube and mixed vigorously with 20 mL of distilled

water. In an empty test tube the green tablet was added with 300 μL of water and mixed 10

seconds to break the tablet. Then, 0.5 mL of the sample or control solution was added and

mixed immediately. After incubation (45 C° for 5 min), the orange tablet was added, and the

solution was mixed immediately. After a second incubation (45 C° for 5 min), the black

tablet was added and the solution was mixed immediately and centrifuged for 5 min at 5000

rpm. Meanwhile, new test tube was labeled and the white tablet with 300 μL of water is

added. The supernatant from the first tube were poured into the new labeled test tube and


37

mixed immediately. After incubation (45 C° for 5 min), the solutions were centrifuged for 5

min at 5000 rpm. Finally, the supernatant was removed and additional 300 μL of water was

added to the tube and mixed thoroughly to break up the pellet. After, 3 mL of scintillation

fluid was added into the tube, which was shaken until the mixture has with a uniform cloudy

appearance. CPM (count per minute) were read on [3H] channel by using (Charm LSC 7600,

Charm Science Inc., USA).

2.8.2. Sulphonamide residues

The sensitivity of Charm II sulfa drug test for honey is set to detect sulphonamide at

10 ng/g or ppb. 5 g of sample were weighted and mixed vigorously with 20 mL of distilled

water. An extraction procedure is required to free sulfa drugs bounded to the sugars in honey

and to eliminate interference from sulfa drug analogs, filtrating the solution followed by SPE

extraction in C18 cartridge. After extraction, a white tablet was added to an empty test tube

than mixed well with 300 μL of water, and followed by the addition of 5 mL of extracted

solution. A pink tablet was then added to the tube and mixed immediately. After incubation

(85C° for 3 min) the solution was centrifuged for 3 min at 3400 rpm. Supernatants were

poured off, fat rings were removed, and test tubes were wiped with swabs to avoid

disturbing the pellet. Finally, 300 μL of water were added into the tube and mixed

thoroughly to break up the pellet. 3 mL of scintillation fluid was added into each tube and

shaken until the mixture has a uniform cloudy appearance. CPM (count per minute) were

read on [3H] channel by using (Charm LSC 7600, Charm Science Inc., USA).

Chapter III- Results and discussion

38



39

3. Results and discussion

3.1. Melissopalynological analysis

Pollen analysis of honey, or mellissopalynology, is of great importance for quality

control. Honey always includes numerous pollen grains (mainly from the plant species

foraged by honey bees) and honeydew elements (like wax tubes, algae and fungal spores)

that altogether provide a good fingerprint of the environment where the honey comes from.

Pollen analysis can therefore be useful to determine and control the geographical and

botanical origin of honeys even if sensory and physicochemical analyses are also needed for

a correct diagnosis of botanical origin. Moreover, pollen analysis provides some important

information about honey extraction and filtration, fermentation (Russmann, 1998),

adulteration types (Kerkvliet et al., 1995) and hygienic aspects such as contamination with

mineral dust, soot, or starch grains (Louveaux et al., 1978).

Multifloral honeys have in their composition percentages of pollen from various

floral species, while monofloral honeys are characterized by honeys obtained mainly from a

single plant species (≥ 45% of the same pollen type), although this value may vary according

to the plant's ability to produce pollen. (Estevinho et al, 2012).

Honey samples EC1 and EC2 from Sidi Belabes region had Cytisus striatus type as

the dominant pollen and accompanying pollen respectively, in fact EC1 and EC2 which were

labeled as eucalyptus and showed low percentages of eucalyptus pollen, Table 11. Cytisus

striatus was also the dominant pollen type of MF1 and MF2, which represented a minimum

of 83.3% to a maximum of 83.8% of the total pollen content, Table 11. Honey samples EF,

from El Bayadh region, had Cytisus striatus type as the dominant pollen for the three

samples, with an average of 79.8%, instead of Euphorbia pollen which were indicated in the

commercial label. Regarding to this pollen type, it can be either the Cytisus striatus type or

another within the same genus, like C. arboreus, C. triflorus, C. purgains, C. pinifolius, C.

fontanesii, C. monspessulanus, C. arboreus, which were previously reported as present in

the areas of the apiaries (Quezel and Santra, 1962). Honey samples J1, J2 and J3 from Ain

Safra region, contained pollen grains from Ziziphus sp. in percentages ranging between 38.4%

and 40.5%. Thus, the pollen of this species is nearly dominant, suggesting that this plant is

the main source of pollen in these honeys, Table 11. Cytisus striatus pollen type was present

in a total of 10 samples and it considered dominant in 7 of them.


40

Table 11. Pollen characteristics of the analyzed honey samples.

Sample Floral origin on

the label D A I

EC1 Eucalyptus sp. Cytisus striatus

type (47.3%)

Brassica napus type

(18.4%)

Eucalyptus sp.

(5.5%);

Sesamoides sp.

(5.9%); Rhamnus

alaternus (13.9%)

EC2 Eucalyptus sp. -

Cytisus striatus type

(39.3%); Brassica

napus type (12.6%);

Rhamnus alaternus

(26.8%);

Eucalyptus sp.

(5.1%);

Sesamoides sp.

(9.1%)

MF1 Multifloral Cytisus striatus

type (83.3%) -

Calina racemosa

(5.0%)

MF2 Multifloral Cytisus striatus

type (83.8%) -

Calina racemosa

(5.0%)

EF1 Euphorbia sp. Cytisus striatus

type (82.3%) -

Centaurea sp.

(4.5%); Brassica

napus type (6.3%)


type (76.9%) -

Centaurea sp.

(5.6%); Brassica

napus type (7.9%)


type (80.2%) -

Centaurea sp.

(6.4%); Brassica

napus type (5.8%)

J1 Ziziphus sp. -

Ziziphus sp.

(39.5%); Eucalyptus

sp. (16.2%); Cytisus

striatus type

(25.8%)

Echium sp. (4.9%)

J2 Ziziphus sp. -

Ziziphus sp.

(40.5%); Cytisus

striatus type

(28.6%)

Eucalyptus sp.

(12.6%); Echium

sp. (3.9%)

J3 Ziziphus sp. -

Ziziphus sp.

(38.4%); Cytisus

striatus type

(26.0%)

Eucalyptus sp.

(15.1%); Echium

sp. (3.4%)

D: Dominant pollen (≥ 45%); A: Accompanying pollen (15% - 45%); I: Important pollen (3% - 15%).

3.2. Physicochemical parameters

3.2.1. Color

The color of honey is closely linked to its botanical origin and is an important

parameter for evaluating honey quality. Honey color is generally related to its sensory

properties such as flavor and odor and can give information on its floral source, mineral

content, and storage conditions. The colorimetric analysis of the honey was performed using

the Pfund scale by the direct reading in the colorimeter. The color ranged from (extra light


41

amber until amber), Table 12, EC1 honey presented the darker color and EF1 and EF3

showed the clearest color. Honey samples EC1 and EC2 showed amber color, with values of

89mm and 88 mm Pfund, respectively, while MF1 and MF2 presented a light amber color,

79 and 77 mm Pfund, respectively. All these results are in accordance with the last study on

multifloral honey samples of Morocco (Chakir et al., 2016), and were similar to those

obtained by (Homrani et al., 2020) on Algerian honeys. J1, J2 and J3 honey samples

showed extra light amber color in which values ranged between 51 and 55 mm Pfund. The

results obtained were very near to those obtained on Citrus and Retama honeys from

Algerian semi-arid region (Homrani et al., 2020). The three EF samples (EF1, EF2, EF3, and

EF4) gave the same color, extra light amber, which were in accordance with those obtained

previously (Homrani et al., 2020).

Table 12. Physicochemical parameters: color, moisture content and conductivity.

Samples Color (mm Pfund) Moisture

content

(%)

Conductivity

(µS.cm- 1

)

EC1 89 ± 0 (Amber)

18 ± 0 410 ± 0.02

EC2 88 ± 0 (Amber)

18 ± 0 410 ± 0.02

MF1 79 ± 0 (Light Amber)

15 ± 0 270 ± 0.01

MF2 77 ± 0 (Light Amber)

15 ±0 300 ± 0.06

J1 55 ± 0 (Extra Light Amber)

15 ± 0 370 ± 0.01


15 ± 0 370 ± 0.01


15 ± 0 370 ± 0.01

EF1 51 ± 0 (Extra Light Amber)

14 ± 0 360 ± 0.01

EF2 52 ± 0 (Extra Light Amber)

14 ± 0 360 ± 0.01

EF3 51 ± 0 (Extra Light Amber) 14 ± 0 360 ± 0.00

3.2.2. Moisture content

Moisture is a parameter related to the maturity degree of honey and temperature. In

the present study, the moisture values varied between 14% (EF1) and 18% (EC1), which

https://www.sciencedirect.com/science/article/pii/S0308814620307329#b0130


42

were within the limit of 20% stablished by the European Community regulations (The

Council of the European Union, 2002), Table 12. EC honey samples have the highest water

content around 18 , which were in accordance with the values found in Hedysarum

coronarium and Eucalyptus honeys from Bejaia region (Ouchemoukh et al., 2006) and

higher than those obtained in Morocco (Chakir et al., 2016). The moisture content of MF

samples were in the order of 15, which were similar to results previously report in

Capparis and multifloral honeys from Bejaia region (Ouchemoukh et al., 2006). However,

when comparing the results with Morocco multifloral honey samples (Chakir et al., 2016),

the latest presented higher water content (17.8) comparing to our samples. In another

hand, the water content of Ziziphus samples, around 15%, are directly in line with the results

previously reported by Algerian Ziziphus honeys (Latifa et al, 2013). The water content of

EF samples was 14%, consistent with what has been previously found in Algerian

Euphorbia honey (Latifa et al, 2013) harvested in the semi-arid region of Algeria.

3.2.3. Electrical conductivity

Electrical conductivity (EC) is closely related to the concentration of mineral and

organic acids and shows great variability according to the floral origin. The sample with

electrical conductivity values higher than 800 μS.cm−1

are considered honeydew honeys.

While those that express values below 800 μS.cm−1

are considered nectar honey or mixtures

of different nectars (Bogdanov, 2011). All analyzed honeys presented values less than 800

μS.cm−1

, ranging between 270 and 410 μs.cm−1

, being considered nectar honeys. EC

samples showed the higher values among our honey samples 410 μS.cm−1

, Table 12. Those

values were within the values found in Algerian honeys (between 410 and 630 μS.cm−1

)

(Djamila B, Paul S, 2010) and less than those found in Moroccan honeys (768.78 μS.cm−1

)

reported by (Chakir et al., 2016). MF honey samples showed values between 270 (MF1) and

300 (MF2) μScm−1

. Hadia et al., (2017) found similar results (100 and 370.5 μS.cm−1

) in

multifloral honey harvested in the east of Algeria. The EC average value for Z honey was

370 μS.cm−1

.The values are lower than those given for Z. lotus of Morocco (673.42 μs.cm−1

)

previously reported (Chakir et al., 2016) and near to those given for Z. lotus of Algeria

(478.25 μS.cm−1

) reported by (Latifa, 2013).

The EC average of EF honeys was 360 μs.cm−1

. Our results are similar to the

findings previously reported by (Latifa, 2013) and lower than those obtained by (Chakir et

al., 2016) on Moroccan Euphorbia samples.












43

3.2.4. pH, free, lactonic and total acidity

Ibrahim Khalil et al., (2012), indicated that honey is naturally acidic regardless of its

geographical origin, which may be due to the presence of organic acids that contribute to its

flavor and stability against microbial spoilage. Nectar honeys usually have low pH values

(3.3 to 4.6). Honeydew honeys have, due to their higher buffering salt content, higher

average pH values (Bogdanov, 1995).

The results obtained in this study show that all the analyzed honeys are acidic and

within the standard limit (Codex Food, 2001), ranging from 4.2 to 5.1, Table 13. EF samples

are the most acidic with (pH=4.37), followed by MF samples (pH=4.44), the lower acidity

was detected in the honey samples from Ziziphus (4.93 in average), while EC honeys

showed values between 4.4 and 4.9. The pH of samples from Algerian semi-arid regions

(Media, Djelfa, El aghouat) was 3.61 to 4.16 and 3.49 to 4.44 (Zerrouk et al. 2011 and

Zerrouk and Bahloul. 2020), respectively.

The pH values of nectar honeys vary between 3.5 and 4.5 and honeydew honeys have

higher average pH values between 4.5 and 5.5 (Gonnet, 1986). We could say that the honeys

studied are of the nectar type.

The acidity of honey is mainly due to gluconic acid (Vaillani and Mary, 1988), which

results from the oxidation of glucose by the enzyme glucosidase from the bee (Russo, 1997).

Rogulja et al., (2009) suggested that honeys with lighter color are characterized by a low

content in organic acids, while darker honeys generally appear richer in acidity. Free acidity

gives information about the origin of honey and influencing its stability (Pataca et al., 2007).

The values obtained for free acidity in our study were between 11and 18.3 meqkg-1

and

between 5.8 and 43.9 meqkg-1

at the two equivalence points (pH=7 and pH=8.3),

respectively. All the honeys analyzed are within the required standard of the Codex

Alimentarius (1998), which is 50 meqkg-1

, indicating an absence of unwanted fermentation

in our samples. The results are also in accordance with previous work carried out on

Algerian honeys. Zerrouk et al. (2011) found values ranging between 14.91 and 40.33

meqkg-1

, while Makhloufi (2010) report values between 17.97– 49.1 meqkg-1

.

Lactonic acidity is considered as an acidity reserve when honey becomes alkaline

(Gonnet, 1982). The values obtained in our lactonic acidity study are between 5.7 and 36.1

meqkg-1

. Total acidity is the sum of free and lactonic acidity, it is a quality criterion, and our

results showed values between 20.1 and 64.7meqkg-1

, and these results indicate that all the

honeys analyzed comply with the standard required by the codex. Our results are higher than


44

those given by Hadia (2020) ranged between 17.12 to 34.29 meqkg-1

on north of Algeria and

are similar to those given on Morocco honeys from semi-arid regions reported by (Chakir et

al 2016) ranged between 11.94–58.03 meqkg-1

Table 13. pH and acidity of the honey samples analyzed.

Sample pH

Free acidity

pH=7

(meqkg-1

)

Free acidity

pH=8,3

(meqkg-1

)

Lactonic

(meqkg-1

)

Total

(meqkg-1

)

EC1 4.4 18.3 ± 0.3 12.2 ± 1.1 17.5 ± 0.6 24.7

EC2 4.9 18.1 ± 0,1 13.0 ± 2.0 15.5 ± 0.7 22.9

MF1 4.2 18.3 ± 0.0 12.7 ± 1.5 17.2 ± 0.5 24.3

MF2 4.6 18.3 ± 0.1 21.9 ± 0.5 31.5 ± 0.4 46.1

J1 4.9 11.5 ± 1.1 22.6 ± 1.6 28.5 ± 0.2 43.2

J2 5.1 12.1 ± 0.8 17.6 ± 0.4 22.8 ± 0.6 35.6

J3 4.8 11.0 ± 0.8 21.7 ± 0.4 35.8 ± 0.5 50.6

EF1 4.4 17.2 ± 0.1 43.9 ± 0.4 27.1 ± 0.1 58.3

EF2 4.3 17.3 ± 0.0 41.6 ± 1.8 36.1 ± 0.3 64.7

EF3 4.4 17.2 ± 0.1 5.8 ± 0.1 5.7 ± 0.1 20.1

3.2.5. Proline

Proline is an important amino acid that originates mostly from the salivary secretions

of Apis mellifera during the conversion of nectar into honey (Bergner and al, 1972). Proline

content is an indication of honey ripeness and, in some cases, sugar adulteration. Some

authors have reported that high concentrations of proline are also typical for honeydew

honeys. Indirectly, proline levels also reflect botanical origin (Cotte and al, 2004). Previous

studies found that the proline content of honey was associated with its floral and

geographical origin (Kečkeš et al, 2013). The proline concentration should be above 0.180

mg/g, lesser values could mean that the honey is possibly corrupted by sugar addition

(Bogdanov. 2002).


45

The studied honey samples have good proline levels (2.2 – 4.7 mg/g), higher than the

minimum limit proposed by Bogdanov et al. (2002), indicating the maturity of the honeys

and absence of adulteration. The proline content in EC ranged between two values 3.4 (EC1)

and 3.6 (EC2) mg/g, two times higher than those found in Algerian honeys given by

(Ouchemoukh and al 2006). As well, the proline average of MF samples was around 3.3

mg/g, ranging between 3.2 (MF1) and 3.4 (MF2) mg/g. These values are two times higher

than those found in Algerian honey given by Ouchemoukh 2006 and similar to those given

by (Latifa, 2013). In the J samples, proline value was around 3.6 mg/g, ranging between 2.7

(J1) and 4.2 (J3) mg/g. Concerning EF, the proline average is around 3.6 mg/kg ranged

between 2.22 (EF1) and 4.7 (EF2) mg/g.

3.2.6. 5-HMF

The presence of 5-HMF in honey result from the slow degradation of fructose which,

in an acidic environment, breaks down and loses three water molecules. This process is

accelerated by heating. The high acidity and water content promote this transformation

(Hadia and Ali, 2017).

HMF is an indicator of the freshness and overheating of honey. According to White

(1978), the level of HMF is a quality criterion of several varieties of food (Nozal et al., 2001)

such as honey, which can provide all the necessary information regarding the heat exposure

of any honey. There are differences between floral and honeydew honeys, between honeys

of various botanical origins and also it depends on the variations in pH and acidity (Hadia

and Ali, 2017). Freshly harvested honey contains virtually no HMF. On the other hand, in

the case of hot storage, this value increases (Bogdanov, 1988; Mendes et al., 1998). The

European legislation (European Honey Directive, 2001) established the limit of 40 mg.kg-1

,

with the exception for honeys from tropical countries or regions where the maximum value

may reach 80 mg.kg-1

.

The results in this study, Table 14, are between 0 and 36.5 mg.kg-1

, being within the

standard required by the European legislation. The HMF average of EC honeys is around 35

mg.kg-1

and are similar to those given by (Djamila B and Paul S, 2010) and beyond those

found in Morocco honeys (between 3.25 and 43.87 mg.kg-1

) by (Chakir et al., 2016). The

HMF average of MF honeys is around 26 mg.kg-1

, while for J samples its around 2 mg.kg-1

.

Those latter are also within those found by (Latifa 2013) in Ziziphus Algerian honey

(between 0 and 6 mg.kg-1

). The HMF average of EF honeys is around 20 mg.kg-1

range

between three values 19.2 (EF1), 18.7(EF2), 21.0 (EF3) mg.kg-1

. Our results were similar to



46

those reported by (Chakir et al., 2016) in Moroccan honey harvested in semi-arid region

(between 12.08 and 20.32 mg.kg-1

).

Table 14. Physicochemical parameters of honey: 5- HMF, diastase and proline.

3.2.7. Diastase activity

Diastase content depends on the floral and geographical origins of the honey.

Diastase enzymes are sensitive to heat and consequently is able to indicate overheating of

the product and the degree of preservation (Ligia et al, 2020).

The results of our honeys were between 8.8 DN and 13.8 DN, they were in

accordance with the minimum of 8 DN established by the European Community Regulation

(The Council of the European Union, 2002). EF samples have the higher values; however J

samples have the lower diastase index. The diastase results are lower than those given in

Moroccan honeys (14.45 DN in average) reported by (Chakir et al, 2016), as well as

Tunisian honeys (17.6 DN in average) reported by (Jilani et al, 2008)

3.3. Sugar analysis

Honey is a supersaturated sugar solution in which major compounds are

monosaccharides (fructose and glucose), which represent about 75% of the sugars found in

Sample

HMF (mgkg-1

)

Diastase (DN)

Proline

(mgg-1

)

EC1 34.2 ± 3.1 9.3 ± 0.1 3.6 ± 0.1

EC2 36.5 ± 2.3 10.1 ± 0.5 3.4 ± 0.0

MF1 25.8 ± 1.8 9.4 ± 0.0 3.2 ± 0.1

MF2 27.7 ± 1.1 9.4 ± 0.4 3.4 ± 0.0

J1 5.9 ± 0.7 9.7 ± 1.0 2.7 ± 0.1

J2 0.0 ± 2.4 8.8 ± 1.1 3.7 ± 0.1

J3 0.0 ± 2.0 12.8 ± 0.4 4.5 ± 0.3

EF1 19.2 ± 1.4 13.8 ± 0.4 2.2 ± 0.2

EF2 18.7 ± 1.7 12.4 ± 0.4 4.7 ± 0.5

EF3 21.0 ± 1.6 12.2 ± 0.4 3.9 ± 0.0




47

honey. The percentage of glucose and fructose for nectar honeys should not be less than

60%, and for honeydew honeys it should be a minimum of 45% (Decree-Law nº 214/2003).

The sugar profile also gives information on the origin of honey, with honeydew honeys

having higher levels of trisaccharides (melezitose or erlose).

All samples under study revealed higher fructose content than glucose, Table 15,

with these two monosaccharides representing more than 88%, which allows to classify them,

in accordance with international legislation, as nectar honeys. The analyzed samples do not

have sucrose which is indicative of no unadulterated honeys. The sugar profile of the

different samples showed a similar composition, with values ranging between 37.8 - 43.4

g/100 g and 29.9 – 36.5 g/100 g for fructose and glucose, respectively. The EF samples,

showed the highest values of glucose and fructose, 43.26 g/100g of fructose and 36.16 g/100

g of glucose, while MF samples showed the lowest values of fructose and glucose, 37.9

g/100 g and 29.9 g/100 g, respectively. The values are in accordance with the Algerian

honey levels of fructose which were found to vary between 33.40 and 48.60 g/100 g and

glucose levels to vary between 26.67 and 38.42 g/100 g (Ouchemoukh et al, 2010).

The sugars in honey are responsible for its viscosity, hygroscopicity and

crystallization. The distribution between the different sugars will provide valuable

information that will allow predicting the rate of crystallization and the stability of the

structure of honey (Pourtallier et al, 1970). Crystallization is occurring naturally in honey

depending on its composition in sugars and moisture and that appears related to the type of

honey. The ratios of F/G (fructose/glucose) and G/H (glucose/moisture) provide information

on predicting the time that a honey sample takes to crystallize. The ratio of fructose to

glucose depends largely on the source of nectar. Many researchers report that the fructose

and glucose ratio have an average value of 1.2 for honey, stating that values greater than 1.3

imply a slow crystallization, above 1.5 indicates that honey does not crystallize and less than

1.1 indicates that crystallization is rapid. This process occurs because glucose is a sugar

more insoluble in water than fructose. The speed at which glucose crystallization occurs also

depends on the G/H ratio. According to the literature (Escuredo et al 2014), the

crystallization of a honey is slow or null when the G/H ratio is less than 1.7 and fast when

the ratio is greater than 2 (Escuredo et al,2014). In Table 15, the samples analyzed at the F/G

ratio have values between 1.2 and 1.3 which can be said that all samples have a slow

tendency to crystallize, and the values of G/H oscillate between 1.7 and 2.6 indicating that

the samples have an average propensity to crystallize.


48

Table 15. Sugar profile, obtained by HPLC-RI, of the studied honey samples (values

expressed in g/100g of honey).

Sample Fructose Glucose Turanose Maltulose Maltose Trealose Rafinose F+G F/G G/H

EC1 39.9 ± 0.6 30.3 ± 0.6 0.6 ± 0.0 3.0 ± 0.6 1.8 ± 0.7 0.6 ± 0.0 N/D 70.2 1.3 1.7

EC2 40.2 ± 0.5 30.6 ± 0.5 0.7 ± 0.0 2.9 ± 0.8 1.5 ± 0.6 0.6 ± 0.0 N/D 70.8 1.3 1.7

MF1 37.8 ± 0.7 29.9 ± 1.1 0.9 ± 0.0 3.8 ± 0.9 2.0 ± 0.2 0.4 ± 0.0 0.8 ± 0.0 67.7 1.3 2.0

MF2 38.0 ± 0.7 29.9 ± 0.0 0.9 ± 0.0 3.6 ± 0.8 1.9 ± 0.1 0.4 ± 0.0 0.8 ± 0.0 67.8 1.3 2.0

J1 40.1 ± 0.7 31.8 ± 0.4 0.2 ±0.0 6.5 ± 0.7 4.8 ± 0.4 1.3 ± 0.3 1.4 ± 0.5 71.9 1.3 2.1

J2 39.9 ± 0.7 31.9 ± 0.3 0.6 ± 0.0 6.6 ± 0.6 4.9 ± 0.4 1.2 ± 0.2 1.5 ± 0.6 71.8 1.3 2.1

J3 40.2 ± 0.3 31.5 ± 0.2 0.6 ± 0.0 5.9 ± 0.4 4.5 ± 0.1 1.5 ± 0.2 1.1 ± 0.0 71.7 1.3 2.1

EF1 43.2 ± 0.6 36.1 ± 1.3 0.9 ± 0.0 3.8 ± 0.9 2.0 ± 0.2 0.4 ± 0.0 0.8 ± 0.0 79.3 1.2 2.6

EF2 43.2 ± 0.6 36.5 ± 1.5 0.9 ± 0.0 3.6 ± 0.2 2.5 ± 0.2 0.5 ± 0.0 0.4 ± 0.1 79.6 1.2 2.6

EF3 43.4 ± 0.7 35.9 ± 0.6 0.9 ± 0.1 3.5 ± 0.0 2.4 ± 0.2 0.5 ± 0.1 0.4 ± 0.1 79.3 1.2 2.6

3.4. Minerals

Honey contains diversified amounts of mineral substances, ranging from 0.02 to

1.03g/100g (White, 1975). Potassium, with an average of about one third of the total, is the

main mineral element (Feller-Demalsy et al., 1989; Gonzalez-Miret et al., 2005). The

amount of different minerals in honey is largely dependent on the soil composition, as well

as various types of floral plants (Anklam 1998). In addition to these factors, the beekeeping

practices, environmental pollution, and honey processing may also contribute to the

diversified mineral content present in honey (Pohl, 2009).

The contents of each mineral found in our honeys expressed in mg/kg are shown in

Table 16. The potassium was quantitatively the most important mineral, 72.93% of total

minerals quantified, having an average content 730.60 mg/kg. Sodium, calcium and

magnesium were present in moderate amounts in the honeys (17.05% and 4.43% and 4.22%

of total minerals, respectively), while cadmium and lead were below the detection limit.

Magnesium content (42.31 mg kg-1

in average) was above the limit 25 mg kg-1

for Mg, iron

(11.4 mg kg-1

in average) and copper (0.33 mg kg-1

in average) concentrations were less than

the maximum limit set by the codex Alimentarius [15 mg kg-1

for iron and of 5 mg kg-1

for

copper] (Yaiche and Khali, 2014; Codex Alimentarius 2001).


49

Lead and cadmium are released into the environment through its use in various

industrial processes, and enters the food chain from uptake by plants from contaminated soil

or water. Moreover, Cd and Pb are considered bioindicators for honey contamination (Licata

et al. 2004). The regulations establish a maximum level of 300 μg kg-1

, recommended by

FAO/WHO/1984 (Al-Eed et al. 2002) while for Cd the European legislation and the Codex

Alimentarius, 2001 fixed a maximum of 0.05 mg kg-1

, nevertheless our results did not reveal

its presence. Z samples showed the highest values of potassium, sodium and calcium

however Euphorbia labeled samples showed the highest values of magnesium, while EC

samples presented the highest values of manganese and MF samples showed the highest

values of iron, lead and cadmium.

Table 16. Minerals contents, obtained by using flame atomic absorption spectrophotometer

(values expressed in mg/100 kg of honey).

Samples Potassium

(mg/kg)

Sodium

(mg/kg)

Calcium

(mg/kg)

Magnesium

(mg/kg)

Manganese

(mg/kg)

Copper

(mg/kg)

Cadmium

(mg/kg)

Iron.

(mg/kg)

Lead

(mg/kg)

J1 979.9±12.

6 285.6±40.2 40.4±4.0 31.8±2.6 0,4±0,0 0.3±0.0 <0.03 8.7±0.1 <0.4

J2 863.7±6.8 243.0±60.3 40.2±5.0 31.9±2.9 0.4±0.0 0.4±0.0 <0.03 8.6±0.4 <0.4

J3 737.2±7.7 84.3±2.2 43.5±9.0 29.9±5.5 0.4±0.0 0.4±0.0 <0.03 8.8±0.5 <0.4

EF1 462.5±27.

1 142.7±5.6 46.6±1.5 50.8±2.5 0.9±0.0 0.3±0.0 <0.03 13.0±1.1 <0.4

EF2 684.4±7.4 229.0±1.0 48.0±2.4 54.2±7.2 0.9±0,0 0.3±0.0 <0.03 12.6±2.2 <0.4

EF3 518.2±2.5 169.3±0.7 32.6±1.4 49.9±5.0 0.9±0,0 0.3±0.0 <0.03 12.8±1.7 <0.4

EC1 937.7±1.4 157.6±2.8 51.1±6.8 47.6±1.2 1.9±0,7 0.3±0.1 <0.03 10.7±0.4 <0.4

EC2 884.2±4.7 177.6±2.8 41.4±6.8 49.3±1.2 0.5±0.1 0.4±0.1 <0.03 11.3±0.4 <0.4

MF1 744.2±3.4 123.8±1,0 85.3±3.1 40.5±2.3 1.8±0,6 0.3±0.0 <0.03 14.9±1.6 <0.4

MF2 494.1±3.5 93.8±1.0 14.7±3.9 37.2±2.3 0.5±0,0 0.3±0.0 <0.03 12.6±1.6 <0.4


50

3.5. Nutritional parameters

Honey is considered of high nutritional value. Its ash content is related to color and

flavor, and it is often observed that honeys with higher ash content are also those that have a

darker color and a stronger flavor (Escuredo et al, 2013). In addition, the ash content also

contributes to the electrical conductivity of honey, with a positive correlation between these

two parameters. The Codex Alimentarius (Codex Alimentarius Commission, 1981) does not

provide values for this parameter. Some studies have shown an average value of 0.17% (w/w)

in honey (Chakir et al, 2011). The results obtained in this study for the ash content, varied

between 0.07 and 0.16%, being within the recommended values for nectar honey, Table 17.

According to Anklam (1998), the proteins in honey are related to plant nectar, bees

enzymes and pollen. The quantity of proteins can vary from 0.1 to 0.7 g/100 g Anklam

(1998). Overheated or long-time stored honeys show a reduction or absence of protein

content (De-Melo et al., 2018).

Table 17. Nutritional values of honey: Ash, energy, proteins and carbohydrates.

Sample

Ash (g/100 g) Protein (g/100 g) Energy (kcal) Carbohydrates

(mg/100g)

EC1 0.16±0.01 0.7 ± 0.00 327±0.0 81.0±0.0

EC2 0.16±0.01 0.6 ± 0.0 326±0.0 80.9±0.0

MF1 0.07±0.01 0.5 ± 0.1 340±0.0 84.5±0.1

MF2 0.11±0.04 0.5 ± 0.2 340±0.0 84.5±0.2

J1 0.13 ± 0.0 0.7 ± 0.0 340±0.0 84.3±0.0

J2 0.13 ± 0.0 0.7 ± 0.0 339±0.0 84.2±0.0

J3 0.13± 0.0 0.7 ± 0.0 341±0.0 84.5±0.0

EF1 0.13 ± 0.0 0.6 ± 0.0 345±0.0 85.7±0.0

EF2 0.13 ± 0.0 0.6 ± 0.1 344±0.0 85.5±0.1

EF3 0.13 ± 0.0 0.6 ± 0.0 344±0.0 85.3±0.0


51

The results obtained in this study vary between 0.5 and 0.7g/100g, Table 17. This

variation can be associated to the type of flora and the diets of the bees (El Sohaimy et al.,

2015). Sample J1 is the richest in protein, with a rate of 0.7 g/100g. This is the sample that

comes mainly from Ziziphus, moderately rich in pollen. Sample MF1 is the poorest in

proteins with a content equal to 0.5g/100g. The range of protein observe in our results are

similar to the results obtained by Ouchemoukh and his collaborators in (2007) who found

values between 0.37 and 0.94 g/100g in the Bejaia (City in the north of Algeria) honeys.

Also, the protein content of most Tunisian honeys was between 0.13 and 0.16 mg / 100g of

honey (Boussaid et al., 2014).

As with the mineral and protein content, there is also no legislation that regulates the

limits for the energy value and carbohydrate content present in the different honeys. The

honey samples studied showed similar values of carbohydrates, ranging from 80.9 to 85.7

g/100g, and of energy value, with values between 326 and 345 kcal, Table17.

3.6. Total phenolics and total flavonoids contents

Polyphenols are a class of important secondary metabolites with multiple phenolic

hydroxyl groups in which the main sources are plant secretions, and includes flavonoids,

phenolic acids, stilbenes, and tannins (hydrolysable and condensed), which are mainly

synthesized by the phenylpropanoid metabolic pathway (Kumar and Goel, 2019). They

possess various pharmacological activities, such as anti-cardiovascular, anti-oxidation, anti-

inflammatory, and anti-tumor effects (Olas B. (2020). Among the structures identified in

honey: phenolic acids (benzoic and cinnamic acids), flavonoids (flavones and flavanones)

are the major compounds detected in variable proportions (Al Mamary et al., 2002 cited in

Yahia Mahammed, 2015). A correlation between the antioxidant activity and total phenolic

content is frequently established in literature [Al, M.L et al, 2009- Aljadi et al, 2004]. The

high levels of flavonoids, phenolic acids, ensure a high level of antioxidants in honey which

is the hallmark of its effect as a natural medical product (Madhavi and Kailash, 2014).

According to Anklam (1998), a careful evaluation of polyphenol content could

probably give an indication of the botanical, geographic and climatic origin of honey and the

conditions of plant sources in the region likewise it allows to differentiate between

honeydew, and nectar honey. Darker honeys are richest in phenolic compounds when

comparing with lighter color honeys (Campus et al., 1983).


52

Table 18. Total phenolic and total flavonoid contents and antioxidant activity of honey

samples.

Sample

Total phenolic

content (mg/GAE.g-1

)

Total flavonoid content

(mg/QE.g-1

)

EC1 1.4 ± 0.0 0.07 ± 0.00

EC2 1.2 ± 0.0 0.05 ± 0.00

MF1 0.8 ± 0.2 0.08 ± 0.01

MF2 0.9 ± 0.2 0.07 ± 0.04

J1 0.7 ± 0.0 0.05 ± 0.00

J2 0.7 ± 0.0 0.03 ± 0.00

J3 0.8 ± 0.1 0.04 ± 0.02

EF1 1.1 ± 0.1 0.06 ± 0.02

EF2 0.7 ± 0.0 0.06 ± 0.00

EF3 0.7 ± 0.0 0.09 ± 0.01

The total phenolic content values obtained in our work vary from 0.7 mg GAE/g

honey (EC1) to 1.4 mg GAE/g honey (EF and J), with an average of 0.9 mg GAE/g honey,

Table 18. Our results are higher than those obtained by Khalil et al., (2012), who reported

values between 0.459 ± 0.0015 mg GAE/g honey for Algerian samples. Douka et al., (2014),

reported values between 1.66 to 4.27 mg GAE /g honey in some honeys from western

Algeria.

The total flavonoid content of honey samples (mg of QE/100 g) varied from 0.03 to

0.09 mg QE/g, Table 18, with the highest levels observed in J honeys. The mean values for

total flavonoids were 0.06 mg QE/g, which were similar to those obtained previously (Khalil

et al, 2012).

3.7. Phenolic compounds by UPLC / DAD / ESI-MSn

Nowadays, new analytical technologies, such as the analysis of the profile of

phenolic compounds, are used to characterize and evaluate the authenticity of honeys

associated with particular botanical origins. The profile of phenolic compounds was

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evaluated by UPLC/DAD/ESI-MSn, after the extraction of these compounds from the honey

samples. The methodology allowed the elucidation of the phenolic compounds by

comparing their chromatographic profile, UV spectrum and mass spectrometry information,

with reference compounds. When standards were not available, structural information was

confirmed with the combination of UV data and MS fragmentations described in the

literature. ESI-MSn in the negative mode was used due to the great sensitivity that this mode

presents in the detection of the different classes of phenolic compounds (Falcão et al, 2013).

Table 19 shows the various compounds identified in each sample, with the respective

retention time, maximum absorbance bands and mass spectrometry information.

Table 19. Phenolic compounds and abscisic acid identified by UPLC/DAD/ESi-MSn in the

honey samples under study.

aConfirmed with standard;

bConfirmed with MS

n fragmentation; Confirmed with references:

cOuchemouck et al.,

2016; dBertoncelj et al., 2011;

eFalcão et al., 2019;

fFalcão et al., 2013;

In this study it was possible to identify nineteen phenolic compounds, which

included nine flavonoids, six phenolic acids, two isoprenoids, one spermidine and one

Nº Compound TR (min) λmax (min) [M-H]- [M-H]

2

1 Benzoic acid derivativeb,c

1.25 284 121, [M+46]-

:167

2 p- Hidroxybenzoic acida,b

1.87 256 137 93

3 Caffeic acida,b

2.07 292, 322 179 135

4 p-coumaric acida,b

2.82 310 163, [M+46]-

:209

5 Salicylic acida,b

6.11 301 137 93

6 Syringetinb

6.38 276 345 161(100), 285(91), 309(21),

327(24)

7 trans, trans- Abscisic

acida,b,d

6.88 265 263 154(100), 153 (69), 220 (36)

8 p- hydroxybenzoic

derivitaveb

7.05 219, 203 199 155(100), 137(20)

9 cis, trans- Abcisic acida,b,d

7.46 265 263 154(100), 153(69), , 220(36)

10 Isorhamnetin rhamnosideb

7.57 254, 354 461 315

11 Pinobanksin-5-methyl-

etherb,f

7.67 287 285 267 (100), 239 (29), 252 (13)

12 Quercetina,b

7.76 256, 370 301 179(100), 151(69)

13 N1, N

5, N

10-tri-p-

coumaroyespermidineb,e

8.31 292, 308 582 462(100), 436(10), 342(7)

14 Pinobanksinb,f

8.33 292 271 253(100), 225(20), 151(10)

15 Kaempferola,b

8.45 269, 345 285 229(100), 151(93), 257(80)

16 Carnosolb

8.92 329 241 (100), 185 (65), 311 (58)

17 Chrysina,b

10 269 253 253(100), 209(49), 225(17)

18 Pinocembrina,b

10.13 290 255 213 (100), 151 (34) 253(100),

271(20)

19 Galangina,b

10.22 265, 300sh, 358 269 269 (100), 241 (61), 227 (20), 151

(20)


54

phenolic diterpene. Among the identified phenolic acids, three are derived from benzoic acid

(benzoic acid derivative, p-hydroxybenzoic acid, salicylic acid and p-hydroxybenzoic acid

derivative) and two are derivatives of cinnamic acid (caffeic acid, p-coumaric acid. Of the

nine flavonoids identified, five belong to the class of flavonols (syringetin, isorhamnetin

rhamoside, quercetin and kaempferol), two to the flavone class (chrysin, galangin), one

flavanone (pinocembrine) and two dihydroflavonols (pinobanksin-5-methyl- ether and

pinobanksin). In addition two isoprenoids, which included two isomers of abscisic acid,

have also been identified (cis, trans- and trans, trans-), as well as carnosol, which is a

phenolic diterpene, and spermidine: N1,N

5,N

10-tri-p-coumaroyespermidine. Among the

compounds identified (Table 20), it can be seen that p-coumaric acid were presented only in

EC samples while kaempferol, pinocembrin and galangin were presented only in J samples.

The trans, trans isomer of abscisic acid was presented in both EC and J samples but it was

presented in high concentration in J honeys than EC honeys. The compounds specific for

one type of sample can be considered as marker compounds for that honey. In Table 20, it

can be seen that the samples that presented the greatest amount of phenolic compounds are

sample EC1 with 202 mg/100 g and with the lowest amount is sample EF3 with 60 mg/100

g. It can be seen that in relation to phenolic acids, the EC1 is the one with the highest

amount of compounds derived from benzoic acid (92 mg/100g) and the EC2 sample stands

out for the acids derivatives of cinnamic acid (58.6mg/100g). These phenolic compounds

were already reported in Algerian honeys (Ouchemoukh et al, 2017). Moreover, it has been

found by Can et al. (2015) that benzoic, caffeic and p-coumaric acids were present in

differing amounts in all unifloral Turkish honeys.

The flavonoids found in honey come from pollen, propolis and nectar, with propolis

being the richest source of flavonoids. Pinobanksin and its derivatives, pinocembrine,

chrysin and galangin are compounds described as propolis derivatives (Falcão et al, 2013;

Tomás et al, 2001). Pinobanksin is present in all samples in exception of EC1 and EC2 and

pinocembrine is present in small amount only in samples J1, J2 and J3, with values ranging

between 0.1-13.5mg/100 g and 003-0.2 mg 100g, respectively, Table 20.

Some authors (Tomás et al, 2001) report that the amount of flavonoids is higher in

honeys harvested during dry seasons with high temperatures and that the darker honeys

contain more derivatives of phenolic acids, while lighter honeys contain more flavonoids

(De-Melo et al, 2017). Abscisic acid (two isomers) is an important phytohormone regulating

plant growth, and has an essential role in multiple physiological processes of plants.

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55

Abscisic acid controls downstream responses to abiotic and biotic environmental changes

(Chen et al, 2020). Its content varied between 8.3 and 20.1 mg/100 g for isomer 1 (trans,

trans- abscisic acid) and 6.2 and 25.7 mg/100 g for isomer 2 (cis, trans- abcisic acid), Table

20. Ouchemoukh and his collaborators, 2017 identified the two isomers in Algerian honeys.


56

Table 20. Quantification of phenolic compounds, expressed in mg/100 g honey.

Compound

EC1 EC2 MF1 MF2 J1 J2 J3 EF1 EF2 EF3

Benzoic acid

derivative 26.3±0.2 19.7±0.1

24.2±1.

3 33.4±0.4 5.9±0.2 4.9±0.0 7.8±0.0 9.7±0.1 14.7±0.2

7.2±0.0

p- Hidroxybenzoic

acid 30.6±1.1 28.0±0.8 8.4±0.3 9.2±1.0 8.6±0.0 10.6±0.7 18.7±0.1 10.6±0.2 17.0±0.4 7.5±0.1

Caffeic acid 5.3±0.6 5.0±0.4 8.9±1.4 10.6±0.5 1.1±0.0 1.0±0.3 1.2±0.3 1.7±0.5 0.01±0.0

0 0.05±0.01

p-Coumaric acid 42.8±0.1 52.5±0.3 - - - - - - - -

salicylic acid 1.9±0.1 2.1±0.2 1.1±0.1 4.0±0.7 2.3±0.1 3.3±0.0 5.0±0.1 1.6±0.2 1.0±0.1 0.1±0.0

Syringetin 14.6±0.1 16.5±1.7 17.3±2.

2 24.9±1.4 9.9±0.2 12.0±1.6 7.1±0.1 35.9±0.1 54.2±0.8 19.6±0.0

trans, trans-

abscisic acid 9.8±0.0 8.3±0.2 - - 20.1±0.6

14.7±0.7

16.3±0.6 - - -

p- hydroxybenzoic

derivitave 35.1±0.4 23.9±0.0

10.8±0.

2 12.2±0.1 3.5±0.1 3.6±0.0 4.0±0.6 2.1±0.0 4.8±0.7 1.4±0.0

cis, trans- Abcisic

acid 9.5±0.0 8.3±0.0 6.2±0.8 9.3±0.4 22.0±0.0 19.3±0.0 25.7±0.1 16.2±0.0 20.3±0.2 8.7±0.0

Isorhamnetin

Rhamnoside 16.1±0.4 10.5±0.9 - - - - - - - -

Pinobanksin-5-

methyl- ether 0.4±0.0 0.2±0.0 0.4±0.0 0.4±0.2 0.4±0.1 0.1±0.0 0.1±0.0 0.4±0.1 0.8±0.1 0.3±0.0

Quercetin 5.2±0.3 1.9±0.0 2.7±0.5 3.9±0.0 9.7±0.2 3.4±0.5 4.5±0.2 9.5±0.2 16.7±1.5 6.7±0.5

N1,N

5,N

10-tri-p-

coumaroyspermidin

e

2.9±0.0 1.1±0.0 0.5±0.1 1.2±0.4 1.1±0.0 2.0±0.2 1.2±0.1 1.1±0.0 2.2±0.2 1.1±0.1

Pinobanksin - - 2.8±0.2 3.9±0.2 0.1±0.0 13.5±0.1 12.8±1.0 11.5±0.0 13.3±0.1 6.0±0.0

Kaempferol - - - - 7.9±0.0 16.5±2.3 3.4±0.1 - - -

Carnosol 1.0±0.0 0.5±0.0 - - 0.7±0.1 0.9±0.1 1.1±0.1 - - -

Chrysin 0.7±0.0 0.9±0.1 0.5±0.0 0.9±0.1 3.4±0.2 2.9±0.2 3.2±0.1 2.4±0.1 3.6±0.1 1.2±0.0

Pinocembrin - - - - 0.03±0.00 0.2±0.0 0.2±0.0 - - -

Galangin - - - - 2.0±0.2 2.6±0.1 3.4±0.3 - - -

Chapter IV- Conclusion and perspectives

57

3.8. Antioxidant activity

3.8.1. DPPH

The scavenging activity of honey samples had been measured by DPPH assay. The

unpaired electron of DPPH forms a pair with hydrogen donated by free radical scavenging

antioxidant from honey and thus converting the purple colored odd electron DPPH to its reduced

form in yellow. The lower the EC50 value the higher the scavenging capacity of honey, because it

requires lesser amount of radical scavenger from the honey to reduce DPPH (Chua et al, 2013).

The values obtained for DPPH in the analyzed samples are represented in Table 21 and ranged

from 0.02 to 0.04 mg/mL, with higher antioxidant activity associated with EC and J honeys and a

lower antioxidant activity associated with EF honeys. The values are correlated with the

concentration of phenolic acids and flavonoids in the samples. Our results are lower than those

obtained in a Moroccan study where the results of DPPH showed EC50 values ranged between

0.245 ± 0.009 mg/mL and 0.832 ± 0.069 mg/mL, meaning that, our honeys have a higher

antioxidant activity than Moroccan samples (El Ghouizi et al, 2021).

Table 21. The antioxidant activity; reducing power and DPPH assay

Sample

Reducing power

(mg/GAE.g-1

)

DPPH

(EC50 mg/mL)

EC1 0.03 ± 0.00 0.02 ± 0.00

EC2 0.04 ± 0.00 0.02 ± 0.00

MF1 0.04 ± 0.00 0.04 ± 0.00

MF2 0.04 ± 0.00 0.03 ± 0.00

J1 0.04 ± 0.00 0.03 ± 0.00

J2 0.04 ± 0.00 0.02 ± 0.00

J3 0.04 ± 0.00 0.02 ± 0.00

EF1 0.04 ± 0.00 0.03 ± 0.00

EF2 0.04 ± 0.00 0.04 ± 0.00

EF3 0.04 ± 0.00 0.03 ± 0.00

3.8.2. Reducing power

Fe (III) reduction is often used as an indicator of electron-donating activity. The presence

of reducing agents in the honey reduced the ferric ions. This reduction is quantified by an

absorbance measurement at 700 nm against a blank, with an increase in absorbance associated

with high reducing power (Mouhoubi, 2016). Table 21 shows the values of the samples evaluated


58

by the reducing power, expressed in equivalents of gallic acid (mg GAE.g-1

). Results of the

reducing power showed that there was no significant difference between our samples observing a

variation between 0.03 and 0.04 mg GAE.g-1

. As described in the literature (Hatami et al, 2014;

Lamuela and Rosa, 2018), it is possible to observe that samples with lower levels of total

phenolic compounds were those that registered lower values of reducing power. Also, the

presence of other non-phenolic compounds such as enzymes (glucose oxidase and catalase) and

non-enzyme materials (vitamins and amino acids) may influence this activity (Aljadi and

Kumaruddin, 2004).

3.9. Cytotoxic potential

The last decade has witnessed an astronomical increase in the amount of research

investigating the role of honey in the treatment of various diseases, including cancer. These

health benefits of honey in treating diverse diseases can be attributed to its various

pharmacologically active constituents, especially flavonoids and phenolic constituents, which

included anti-inflammatory, antioxidant, antiproliferative, antitumor, antimetastatic and

anticancer Candiracci et al, 2012; Samarghandian et al, 2011).

The cytotoxicity of the Algerian honeys was evaluated in four human tumor cell lines

(AGS-gastric adenocarcinoma, CaCo colorectal adenocarcinoma, MCF-7 breast adenocarcinoma,

NCI H460- lung carcinoma) and a non-tumor cell line, Vero (African green monkey kidney). All

the studied extracts inhibited the growth of the mentioned tumor cell lines. MF1 gave the highest

cytotoxicity, followed by EF1, Table 22, presenting the lowest GI50 values against the tested tumor

cell lines. The AGS cell line was the most sensible to the studied samples in the average, the MF1

extract was the most active (GI50 8.1μg/mL; an excellent GI50 value in comparison with Portuguese

Propolis extracts for example (Ricardo et al 2014). This activity could be related to the chemical

composition of those samples. From the analysis of Table 20, it can be observed that samples EF1

and MF1 have significant concentrations of total phenolic and total flavonoids compounds. The

EF3 sample showed the highest GI50 values for all the tested tumor cell lines with an average (375

μg/mL). This fact could be explained by its poor phenolic composition. These results can be

explained also by the level of hydrogen peroxide of these samples. Hydrogen peroxide was

reported to be responsible for the proliferative effect of honey in cancer cells (A. Henriques et al,

2006).

Despite the high cytotoxicity displayed by most of the honey samples against tumor cell

lines studied, the samples also showed toxicity for non-tumor (normal) cell line, however they

reporting higher GI50 values when compared to tumor cell lines.

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59

Ricardo and his collaborators in 2014 found that total flavonoids were positively

correlated (R2 values higher than 0.5) with the cytotoxicity. However, the cytotoxicity was not

correlated (R2 values lower than 0.5) with flavonols, dihydroflavonols, and flavonoid esters.

The present data highlight the high cytotoxicity of Algerian honeys against tumor cell

lines, being in agreement with Siti Noritrah et al. 2019, who reported a marked activity of

Malaysian honey against human lung adenocarcinoma epithelial cell line (A549). As well, our

results were similar to those obtained by Hamada et al, 2019 on Moroccan and Palestinian

honeys rom different regions.

Table 21. Cytotoxicity potential (GI50 values, µg/mL). and anti-inflammatory activity (CI50 values,

µg/mL).

3.10. Anti-inflammatory activity

Inflammation usually occurs when infectious microorganisms such as bacteria, viruses

or fungi invade the body, reside in particular tissues and/or circulate in the blood (Artis and

Spits, 2015; Isailovic et al,2015). Inflammation may also happen in response to processes such

as tissue injury, cell death, cancer, ischemia and degeneration (Artis and Spits, 2015, Lucas et

al, 2006). Mostly, both the innate immune response as well as the adaptive immune response

are involved in the formation of inflammation.

The anti-inflammatory activity of our honey samples was assessed using the mouse

macrophage (RAW 264.7) cell line. All honey extracts under study showed anti-inflammatory

capacity, with IC50 values between 8 and 400 µg/mL. The highest activity was observed for

sample J2, followed by the samples J1 and EC1, with an IC50 value of 9 µg/mL. In opposite the

MF1 sample showed the highest IC50 values for the tested cell line more than 400 μg/mL Table

21. This fact could be explained by its poor phenolic composition. This is the first time, to the

Cell

lines

GI50

EC1 EC2 MF1 MF2 EF1 EF2 EF3 EF4 J1 J2 J3

CaCo 13.7±0.2 176±16 151±7 8.1±0.2 194±17 >400 >400 22.1±0.3 62±1 228±11 71±7

AGS 60±5 9±1 193±8 48±2 194±17 >400 >400 22.1±0.3 62±1 228±11 72±7

MCF-7 383±23 371±3 65±2 281±18 249±25 >400 >400 271±2 >400 >400 >400

NCl-

H460 328±5 283±4 168±10 359±5 163±10 221±23 300±31 212±10 >400 >400 >400

VERO 254±7 245±8 >400 302±21 >400 >400 >400 237±7 >400 >400 >400

RAW 9.5±0.2 43±1 >400 12.7±0.1 57±3 82±4 150±4 9±1 9±1 8±1 8.5±0.3


60

best of our knowledge, that the effects of Algerian honey extracts on anti-inflammatory

activity have been evaluated in vitro.

3.11. Screening of antibiotics residues

Tetracyclines are commonly applied in the treatment of many bacterial infections of the

digestive system, the respiratory system and the skin. Also they are used as a growth stimulant in

animals, in some countries its commonly use as additive in animal feed. The large-scale

application of tetracyclines carries the risk of their residues appearing in food. For other side,

sulphonamides has been used for treatment of American foulbrood (Paenibacillus larvae subsp,

larvae) a deadly disease to honeybees. In 1940, sodium sulfathiazole was registered in the USA

for the control of AFB (Moreno et al, 2009. In some countries outside Europe the use of

tetracyclines, sulphonamides and other antibiotics is still legalized for the treatment of American

foul brood (Reybroeck, 2002). Oxytetracycline is currently the only antibiotic registered for use

by Canadian beekeepers to treat American foulbrood (AFB), a highly contagious bacterial

disease of larvae, difficult to eradicate, caused by the rod-shaped bacteria Paenibacillus larvae).

In Europe this is an illegal practice because ubiquitous administration of antibiotics may cause

bacteria to become resistant to many drugs and spread antibiotic-resistant strains of bacteria

(Żaneta et al, 2011). Antibiotic resistance has become a major concern due to overuse of

antibiotics, leading to difficult to treat infections in humans and animals, with increased

morbidity and mortality (Lekshmi et al, 2017). Because of that, the presence of residues of

antibiotics in European honey is not permitted.

Table 22. Residues screening using CHARM II.

Sample Sulfonamide (10 ppb) Tetracycline (15 ppb)

EC1 2205 Negative 2635 Negative

EC2 2183 Negative 2575 Negative

MF1 1525 Positive 2530 Negative

MF2 1751 Negative 2560 Negative

J1 2408 Negative 1980 Negative



EF1 1050 Positive 1663 Negative

EF2 2267 Negative 2523 Negative

EF3 1475 Positive 1677 Negative


61

The charm II test is a screening test used for different food matrix such as meat and milk.

This has been adapted for honey testing (Bogdanov, 2003), allowing the detection of many

antibiotics (penicillin, tetracycline, macrolides, sulfonamides, and aminoglycosides) by an

immunocompetition reaction between the molecule to be sought and a molecule marked with

C14 or H3 (Audigie et al, 1995). The results of the residues screening for sulfonamides and

tetracyclines in our samples are summarized in Table 22. Out of this monitoring and screening

data it could be concluded that the frequency of antibiotics residues agents in Algerian honeys

from local beekeepers is very low, but still a concern if international trade is to be considered. In

case of tetracycline residues all the results were negative; on the other hand, three of our samples

(MF1, EF1, EF3) showed positive results for Sulfonamide residues.


62

Chapter IV- Conclusion and

Future Perspectives


63

Conclusion

The results of the melissopalynological analysis show that the honey samples analyzed

contain a great diversity of pollen grains, with no elements of honeydew being identified,

which allows us to conclude that these are nectar honeys. Ten types of pollen were identified,

Cytisus striatus pollen were the most abundant, being present in all samples with percentages

between 26.0 % and 83.8 %, with samples EC1 (region of Sidi Belabes), MF1 and MF2

(region of Sidi Belabes) classified as monofloral Cytisus striatus honey. Although samples J1,

J2 and J3 were not consider monofloral, they showed high percentages of Ziziphus pollen

(greater than 39.5 %). The remaining samples were classified as multifloral. The results of the

melissopalynological analysis seem to indicate that no samples of honey really correspond to

the beekeeper classification. Thus, although food security is not at stake, the need to create

additional mechanisms to ensure the authenticity of this type of food product becomes

imperative.

There was a significant difference in color remarked between all studied samples of

honey ranged between amber, light amber and extra light amber. Changes in color might be

attributed to the beekeeper’s interventions and different ways of handling the combs such as

using of old honeycombs, contact with metals and exposure to either high temperatures or light.

The higher Pfund and color intensity values might indicate higher phenolic compounds and

flavonoids. The moisture content of the honey samples was within the limits established by the

legal requirements, that is, less than 20%, which allows us to conclude that the honey will have

been extracted with the appropriate degree of maturation. Regarding electrical conductivity,

the honey samples analyzed showed values between 270 and 410 μS.cm-1

. In general, all

samples showed conductivity values below 800 μS.cm-1

, which means that confirms the

samples as nectar origin. The values established by Codex Alimentarius clearly confirm our

results. The pH values were between 4.2 and 5.1 which again point out for the nectar origin.

The values of free acidity were between 5.8 and 45.0 meq.kg-1

, being below the 50.0 meq.kg-1

stipulated in the Codex Alimentarius, indicating the absence of undesirable fermentation

processes for the quality of honey. The evaluation of the 5-HMF content and the diastase index

provides important information about the quality of the honey, namely about the occurrence of

heat treatments or inadequate storage conditions. The results were in accordance with the

European legislation, ranging between 0 and 36.5 mg.kg-1

. Regarding diastase, the results

ranged between 8.8 and 13.3 DN, being within the quality legal requirements. Honey samples

presented high proline levels (2.2–4.7 mg/kg), indicating a good maturity of the honeys and

absence of adulteration. For the proteins, the values varied between 0.5 and 0.7 mg/100 g. This


64

variation can be attributed to the type of flora and the diets of the bees.

All samples showed higher fructose than glucose content, with these two

monosaccharides representing more than 89%, allowing the classification of the honeys as nectar

honeys. The presence of sucrose was not detected, indicating unadulterated honeys.

Concerning the mineral content, the potassium was found to be the most important

mineral (73% of total minerals quantified), followed by sodium, calcium and magnesium, with

17%, 4.4% and 4.2% of total minerals, respectively. Cadmium and lead where below the limit of

detection.

The determination of the total phenolic compounds content by the Folin-Ciocalteau

method showed values between 0.7 mg GAE/g honey (EF and J) and 1.4 mg GAE/g honey (EC).

The total flavonoid content of honey samples varied from 0.03 to 0.09 mg QE/g honey, with the

highest levels observed in jujube honeys. The scavenging activity of the honeys was evaluated by

DPPH assay, with results ranging 0.02 to 0.04 mg/mL, with higher antioxidant activity associated

with EC and J honeys and a lower antioxidant activity associated with EF honeys. Regarding the

reducing power activity, results showed that there was no significant difference between our

samples observing a variation between 0.03 and 0.04 mgGAE.g-1

.

The analysis of the phenolic compounds profile was performed by UPLC/DAD/ESI-MSn,

where was possible to identified nineteen phenolic compounds (six phenolic acids and nine

flavonoids), two isoprenoid compounds (abscisic acid isomers), one phenolic diterpene (carnosol)

and one spermidine (N1, N

5, N

10-tri-p-coumaroyespermidine). The honey samples analyzed

showed a similar phenolic composition, in which the different compounds are present in almost

all samples, with some differences in their concentrations. Among the compounds identified, it

can be seen that p-coumaric acid, syringetin as well benzoic acid are those that were detected in

most samples in higher concentrations, followed by the two isomers of abscisic acid (cis, trans-

and trans, trans- isomers). Sample EC1 presented the highest quantity of phenolic compounds,

with 202 mg/100 g, while EF3 showed the lowest amount with 59.85 mg/100 g.

The anti-inflammatory activity of the samples was assessed using the mouse macrophage

(RAW 264.7) cell line. All honey extracts under study showed anti-inflammatory capacity, with

GI50 values between 8 and 400 µg/mL. The highest activity was observed for sample J2, followed

by the samples J1 and EC1, with an GI50 value of 9 µg/mL. The cytotoxicity of the Algerian

honeys was evaluated in four human tumor cell lines (AGS-gastric adenocarcinoma, CaCo-

colorectal adenocarcinoma, MCF-7 breast adenocarcinoma, NCI H460- lung carcinoma) and a

non-tumor cell line, Vero (African green monkey kidney). All the studied extracts inhibited the

growth of the mentioned tumor cell lines. MF1 gave the highest cytotoxicity, followed by EF1.


65

The use of antibiotics in beekeeping is an illegal practice in Europe because ubiquitous

administration of antibiotics may cause bacteria to become resistant to many drugs. The

frequency of antibiotics residues in Algerian honeys from local beekeepers is very low. For

tetracycline residues, results were negatives while, three of the samples (MF1, EF1, EF3) showed

positive results of sulfonamide.

Future perspectives

This study concerned the characterization and evaluation of samples from semi-arid

regions in Algeria, and the verification of its compliance with the established legal standards. In

the continuation of this work some recommendations for future research are given below:

It would be important to confirm these results by analyzing more samples of these honeys,

specially Cytisus striatus, considering that this is the first time that this type of mono

flower honey from Algeria has been studied;

A statistical analysis must be applied to obtain the correlation between different

parameters and the influence of each parameter to another;

Identify potential floral markers of the honeys of Cytisus striatus, namely through the

evaluation of the profile in volatile compounds;

A comparison between Algerian honeys and Portuguese honeys with same floral source

should be studied.


66

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Appendix

83

Chapter VI- Appendix

Appendix

84

Appendix

Attached are two abstracts that resulted from two panel communications:

Seloua Kaid, Soraia I. Falcão, Andreia Tomás, Ziani Kaddour,

Miguel Vilas-Boas. Physico-

chemical evaluation of Algerian honeys: Eucalyptus, Jujube, and Spurge, multifloral. NPA

(Natural Products Application: Health, Cosmetic and food), Online Edition 4-5 Feb 2021.

Seloua Kaid, Soraia I. Falcão, Andreia Tomás, Ziani Kaddour,Miguel Vilas-Boas.

Characterization of Algerian honeys by phenolic compounds LC-DAD-ESI/MSn analysis:

Eucalyptus, Jujube, Spurge and multifloral.7 PYCHEM (Portuguese Young Chemists Meetings),

20-22 May 2021 Bragança

Appendix

85

PHYSICO-CHEMICAL EVALUATION OF ALGERIAN HONEYS: EUCALYPTUS, JUJUBE,

SPURGE AND MULTIFLORAL

Seloua Kaid,1,2

Soraia I. Falcão,1, Andreia Tomás,

1 Ziani Kaddour,

2 Miguel Vilas-Boas

1*

1 Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de

Santa Apolónia, 5300-253 Bragança, Portugal; 2 Laboratory of Biotoxicology, Pharmacognosy

and Biological Valorization of Plants, Department of Biology, Taher Moulay University of Saida,

Saida, 20000, Algeria. *[email protected]

Arid and semi-arid zones represent nearly two-thirds of Algerian area. The immensity of these

territories and the absence of systematic studies of the bee flora, make honeys from these regions

poorly studied and poorly understood. The aim of the present study was to evaluate the quality of

semi-arid Algerian honeys and verify its compliance with the established honey standards. For

that, ten samples with different botanical and geographical origin, Eucalyptus (EC), Jujube (J),

Euphorbia (EF) and multifloral (MF), were analyzed regarding the following physicochemical

parameters: moisture, color, pH, free acidity, electrical conductivity, hydroxymethylfurfural

(HMF), diastase index and proline. Concerning the moisture content, the samples presented

values below the 20 % allowed by European Community regulations, ranging from 13.6% (EF)

and 18.3% (EC). Eucalyptus honeys showed a darker color when comparing to the other samples

All honey samples presented conductivity values lower than 0.8 ms.cm−1

, ranging between 0.27

(MF) and 0.41 (EC) ms.cm−1

which are in accordance with the standard results for nectar honeys.

The honeys pH values varied between 4.2 (MF) and 5.1 (J) with an average value equal to 4.6.

For free acidity, tested at pH 8.3, the values where between 12.2 meq.kg-1

(EC) and 43.9 meq.kg-1

(EF). The HMF levels observed for the samples had a minimum of 0.53 (J) and a maximum of

36.5 (EC) mg.kg-1

, while diastase values ranged between 8.8 DN and 14.3 DN, being in

accordance with the required by the European legislation (<40 mg.kg-1

and not less than 8 DN).

For proline, the values ranged between 2.2 and 4.7 mg/g indicating the maturity of the honeys

and absence of adulteration. Generally, the samples were found to meet the requirements of the

international honey standards and were within those found in previous studies about

physicochemical properties of Algerian and Moroccan honeys [1].

References

[1] C. Makhloufi, J.D., Kerkvliet, G.R., D’albore, A. Choukri, R. Samar, Apidologie, 41 (2010)

509.

Acknowledgments

The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for

financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). National

funding by FCT- Foundation for Science and Technology, through the institutional scientific

employment program-contract with Soraia I. Falcão.

Appendix

86

Appendix

87

Characterization of Algerian honeys by phenolic compounds LC-DAD-ESI/MSn

analysis: Eucalyptus, Jujube, and Spurge and multifloral

Seloua Kaid,1 Soraia I. Falcão,

1 Andreia Tomás, Ziani Kaddour,

2 Miguel Vilas-Boas

1*

1 Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de

Santa Apolónia, 5300-253 Bragança, Portugal; 2 Laboratory of Biotoxicology, Pharmacognosy

and Biological Valorization of Plants, Department of Biology, Taher Moulay University of Saida,

20000 Saida, Algeria. *[email protected]

Honey is a complex hive product produced by Apis mellifera bees, composed mainly by

carbohydrates and containing small amounts of other constituents such as minerals, proteins,

vitamins, organic acids, phenolic compounds, enzymes, and other phytochemicals [1]. The

quality of a honey is correlated with its chemical composition and botanical origin. The phenolic

profiles of honeys are determined by their phyto-geographical origin(s), and by the climatic

conditions of the collection site [2]. Thus, identification and quantification of the phenolic

compounds present in honey is of great interest for its origin assessment.

The aim of this research is to determine the phenolic composition of selected honeys collected

from the semi-arid region of Algeria. For that, eleven honey samples, including three from

eucalyptus, four from spurge, three from jujube and two from multifloral botanical origin. The

phenolic compounds were extracted and analyzed trough liquid chromatography coupled to diode

array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS) operating in

negative ion mode. The analysis of the UV spectra together with the molecular ion identification

[M-H]- and MS

n fragmentation allowed the identification of twenty-two phenolic compounds,

among which the most abundant were the abscisic acid isomers (m/z 263), p-hydroxibenzoic acid

(m/z 137), p-coumaric acid (m/z 163), quercetin (m/z 301) and pinobanksin (m/z 271. The

phenolics identified varied quantitatively depending on the botanical origin, with Eucalyptus

honey showing the highest content of phenolic compounds.

References

[1] J. Bertoncelj, T. Polak, U. Kropf, M. Korošec, T. Golob, Food Chemistry, 127 (2011) 296.

[2] S. Ouchemoukh, N. Amessis-Ouchemoukh, M. Gómez-Romero, F. Aboud, A. Giuseppe, A.

Fernández-Gutiérrez, A. Segura-Carretero, LWT-Food Science and Technology, 85 (2017) 460.

Acknowledgments

The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for

financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). National

funding by FCT-Foundation for Science and Technology, through the institutional scientific

employment program-contract with Soraia I. Falcão.

Chapter V- References

88

quality evaluation of algerian honeys: eucalyptus, jujube

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